Phospholipid compounds and formulations

ABSTRACT

The present disclosure provides phospholipid-containing compounds, pharmaceutical compositions and microspheres that exhibit high affinity for mineralized metals. The present disclosure also provides strategies for using said compounds, compositions and microspheres in the treatment of nephrolithiasis or kidney stone disease, and methods of manufacturing and preparing said compounds and compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/775,253, filed Jan. 28, 2020, which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Microspheres with fluid cores enclosed by thin shells made up of lipid,proteins, and/or sugars have been used in medical and therapeuticproducts. These micron-scale, gas-core particles, sometimes referred toas microparticles, have been administered systemically to increase theinformation content of echocardiogram. Microspheres can be particularlyuseful for opacifying the heart's left ventricle, allowing theechocardiogram to more precisely reveal details of aortic valveoperation. The category of microsphere products designed to be used inthis manner for disease diagnosis are often referred to as ultrasoundcontrast agents (UCAs). UCA usage, while making use of mechanical energysources (e.g. the ultrasound producing the echocardiogram) and in whichan associated microsphere mechanical response invariably occurs,typically do not rely, for their function, on tissue effects from themicrosphere mechanical response, and in fact are often engineered tominimize tissue effects.

Microspheres can also be used in therapies in which the microspheremechanical response, with either direct or indirect associated tissueeffects, is central. For example, it has been suggested that for certaincancers, microspheres can be used for targeted delivery ofchemotherapeutic agents. For the targeted delivery, microsphere designgenerally entails a combination of tumor-specific ligands in the shellstructure and fluid-phase chemotherapeutic agents in the core. Afteradministration, the tumor-specific ligands in the shells are expected tomake microspheres accumulate in and around the tumor. A mechanicalenergy source can then be used to break open the microspheres, releasingthe chemotherapeutic agents from the core.

Microsphere mechanical effects can also be used in therapies forpathological biomineralizations, as described in U.S. Publication No.2013/0123781, which is incorporated by reference in its entirety herein.In these therapies, the microspheres are designed to produce pressureeffects resembling those of shock wave lithotripsy, in which powerfulshock waves are focused onto a stone in the kidney or ureter. Inshockwave lithotripsy, the focused high-intensity pressure waves, withpeak pressures above 100 megapascals, bring about cavitation effectsthat progressively erode, pit and fragment the stone. Inmicrosphere-based lithotripsy, microspheres accumulating on the stonesurface can exhibit cavitation effects with similar or greater pressuresthan those in conventional shockwave lithotripsy, but with inputmechanical energies some two orders of magnitude lower (Pishchalnikov etal., 2018); less intense input acoustic energies, compared toconventional shockwave lithotripsy, can have a variety of advantagesboth from a clinical workflow and patient outcome standpoint.Microsphere accumulation on stone surfaces can be facilitated by theincorporation of bisphosphonate-like components into the microsphereshell.

The mechanical effects of microspheres can be used in treatment of avariety of medical conditions involving an abnormal or obstructive mass,such as kidney stones, urinary stones, biliary stones, blood clots,fibroids, cancerous tumors, and atheromatous plaques. The treatmentmethods allow minimally invasive treatments of the medical conditions bydestroying or reducing this mass without injury to healthy tissues andminimizing the pain, discomfort, and risks associated with surgical orother invasive treatments.

Given the wide range of therapeutic application of microspheres, effortshave been made to develop improved components for therapeuticmicrospheres. Despite the efforts, there remains a need in the art forimproved phospholipid compounds that are safe and therapeuticallyeffective. Additionally, it is important to develop phospholipidcompounds that can be produced in large quantities and high qualitiesfor therapeutic and commercial use of therapeutic microspheres.

SUMMARY

The present disclosure provides compounds, pharmaceutical compositions,and microsphere particles (or microspheres or particles) that exhibithydrophobicity and a high affinity for calcium and other metals inmineralized forms, including biomineralizations. Furthermore, thepresent disclosure provides methods of manufacturing and preparing thecompounds, compositions and microsphere particles and methods of usingthem for treatment of medical conditions involving an abnormal orobstructive mass, e.g., nephrolithiasis or kidney stone disease. Themethods described herein allows production of microspheres of highqualities in large scales, allowing therapeutic and commercial use ofthe therapeutic microspheres.

Accordingly, in a first aspect, the present disclosure provides acompound of Formula IV

or a salt, isomer, or salt of an isomer thereof, wherein:

p is from 10 to 30;

q is from 1 to 100;

C is selected from the group consisting of:

B is selected from the group consisting of: a covalent bond and an ethylgroup;

A is selected from the group consisting of: a covalent bond, acyl,acylamino, aminoacyl, acyloxy, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyloxy,aminosulfonylamino, aminosulfonyl, amidino, and carboxy ester; and

X is selected from the group consisting of: hydrogen, silyl, acyl,aminoacyl, thioacyl, aminocarbonyl, aminoacyl carbonyloxy,aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl,substituted sulfinyl, carboxy ester, phthalimido, SO₃H and PO₃H.

In some embodiments, p is 14 or 16, and q is from 38 to 50.

In some embodiments, the compound of Formula IV is the compound ofFormula I

or a salt, isomer, or salt of an isomer thereof, wherein:

n is from 10 to 30, and

m is from 1 to 100.

In some embodiments, n is 14 or 16 and m is from 38 to 50.

In some embodiments, the compound of Formula I is the compound ofFormula Ia

or a salt, isomer, or salt of an isomer thereof thereof.

In some embodiments, the compound of Formula I is the compound ofFormula Ib

In the second aspect, the present disclosure provides a pharmaceuticalcomposition, comprising:

a compound of Formula IV

or a pharmaceutically acceptable salt, isomer, or salt of an isomerthereof;

wherein:

p is from 10 to 30,

q is from 1 to 100,

C is selected from the group consisting of:

B is selected from the group consisting of: a covalent bond and an ethylgroup;

A is selected from the group consisting of: a covalent bond, acyl,acylamino, aminoacyl, acyloxy, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyloxy,aminosulfonylamino, aminosulfonyl, amidino, amide, and carboxy ester;and

X is selected from the group consisting of: hydrogen, silyl, acyl,aminoacyl, thioacyl, aminocarbonyl, aminoacyl carbonyloxy,aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl,substituted sulfinyl, carboxy ester, phthalimido, OH SO₃H and PO₃H.

In some embodiments, p is 14 or 16, and q is from 38 to 50.

In some embodiments, the composition further comprises:

a compound of Formula II

or a pharmaceutically acceptable salt, isomer, or salt of an isomerthereof;

a compound of Formula III

or a pharmaceutically acceptable salt, isomer, or salt of an isomerthereof;

wherein:

t is from 10 to 30,

y is from 1 to 100, and

z is from 10 to 30.

In some embodiments, t is 14 or 16, z is 14 or 16, and y is from 38 to50.

In some embodiments, the compound of Formula IV is a compound of FormulaI

or a pharmaceutically acceptable salt, isomer, or salt of an isomerthereof, wherein:

n is from 10 to 30, and

m is from 1 to 100.

In some embodiments, n is 14 or 16, and m is from 38 to 50.

In some embodiments, the compound of Formula I is the compound ofFormula Ia

or a pharmaceutically acceptable salt, isomer, or salt of an isomerthereof.

In some embodiments, the compound of Formula Ia is the compound ofFormula Ib

In some embodiments, the composition further comprises a fluid with anormal boiling point less than 30° C. and, optionally, at least onepharmaceutically acceptable excipient. In some embodiments, the fluid isa gas at body temperature. In some embodiments, the fluid has lowsolubility in aqueous solutions. In some embodiments, the fluid is air,nitrogen, argon, carbon dioxide (CO₂), sulfur hexafluoride, afluorinated C₁₋₆ alkane, or a combination thereof. In some embodiments,the fluorinated C₁₋₆ alkane is selected from octafluoropropane,n-decafluorobutane, and dodecafluoropentane.

In some embodiments, the composition comprises 0.01-5 mol % of thecompound of Formula IV or Formula I, or a salt, isomer, or salt of anisomer thereof. In some embodiments, the composition comprises 5-9.9 mol% of the compound of Formula II, or a salt, isomer, or salt of an isomerthereof. In some embodiments, the composition comprises 80-95 mol % ofthe compound of Formula III, or a salt, isomer, or salt of an isomerthereof. In some embodiments, the composition comprises no more than 5mol % of the compound of Formula IV or Formula I, or a salt, isomer, orsalt of an isomer thereof. In some embodiments, the compositioncomprises no more than 10 mol % of the compound of Formula II, or asalt, isomer, or salt of an isomer thereof. In some embodiments, In someembodiments, the composition comprises no more than 95 mol % of thecompound of Formula III, or a salt, isomer, or salt of an isomerthereof.

In some embodiments, the composition comprises the ammonium salt of thecompound of Formula IV or Formula I. In some embodiments, thecomposition comprises the ammonium salt of the compound of Formula II.

In some embodiments, the molecular weight of the compound of Formula IVor Formula I is 1,500 to 5,000 Daltons. In some embodiments, themolecular weight of the compound of Formula II is 1,500 to 5,000Daltons. In some embodiments, the molecular weight of the compound ofFormula III is 500 to 2,000 Daltons.

In some embodiments, the molar ratio of the compound of Formula IV orFormula I to the compound of Formula II ranges from 1:100 to 1:1. Insome embodiments, the molar ratio of the compound of Formula II to thecompound of Formula III ranges from 1:20 to 1:8.

In some embodiments, the compound of Formula Ia is a compound of FormulaIb

the compound of Formula II is a compound of Formula IIb

the compound of Formula III is the compound of Formula IIIa

and

the composition comprises the fluid, wherein the fluid isn-decafluorobutane.

In some embodiments, the average molecular weight of the compound ofFormula Ib is about 3200 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula IIb is about 2800 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIIa is about 790 Daltons.

In some embodiments, the composition comprises 0.01-5 mol % of acompound of Formula Ib. In some embodiments, the composition comprises5-9.9 mol % of a compound of Formula IIb. In some embodiments, thecomposition comprises 80-95 mol % of the compound of Formula IIIa. Insome embodiments, the composition comprises no more than 5 mol % of acompound of Formula Ib. In some embodiments, the composition comprisesno more than 10 mol % of a compound of Formula IIb. In some embodiments,the composition comprises no more than 95 mol % of the compound ofFormula IIIa.

In some embodiments, the molar ratio of the compound of Formula Ib tothe compound of Formula IIb ranges from 1:100 to 1:1. In someembodiments, the molar ratio of the compound of Formula IIb to thecompound of Formula IIIa ranges from 1:20 to 1:8.

In some embodiments, the composition is capable of forming microspheresin the presence of water. In some embodiments, the composition comprisesmicrospheres. In some embodiments, the microspheres comprise thecompound of Formula Ib, the compound of Formula IIb, and the compound ofFormula IIIa.

In some embodiments, the microspheres have a mean diameter of about 0.5micron to about 10 microns. In some embodiments, the microspheres have amean diameter of about 1 micron to about 5 microns.

In some embodiments, the composition comprises trehalose and PLASDONEK12 as excipients. In some embodiments, the composition comprisesmicrospheres existing as lyophilized dry powder or as water-freeconcentrate.

In another aspect, the present disclosure provides a method ofmanufacturing microspheres, the method comprising:

(a) preparing a formulation for making microspheres comprising:

-   -   a compound of Formula I

-   -   or a salt, isomer, or salt of an isomer thereof;    -   a compound of Formula II

-   -   or a salt, isomer, or salt of an isomer thereof;    -   a compound of Formula III

-   -   or a salt, isomer, or salt of an isomer thereof;    -   optionally, at least one pharmaceutically acceptable excipient;        and    -   water,    -   wherein:    -   n is from 10 to 30,    -   m is from 1 to 100,    -   t is from 10 to 30,    -   y is from 1 to 100,    -   z is from 10 to 30, and    -   X is selected from the group consisting of: hydrogen, silyl,        acyl, aminoacyl, thioacyl, aminocarbonyl, aminoacyl carbonyloxy,        aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl,        substituted sulfinyl, carboxy ester, phthalimido, OH, SO₃H and        PO₃H.

In some embodiments, the method further comprises:

(b) combining the formulation of step (a) with a fluid with a normalboiling point of less than 30° C. in a vessel; and

(c) agitating the vessel containing the formulation and fluid from step(b) thereby obtaining microspheres.

In some embodiments, the method further comprises:

(d) processing the microsphere solution from step (c) to lengthen itsshelf life or expand the range of environmental conditions for storage.

In some embodiments, the method further comprises:

(e) stoppering the vessel comprising the microspheres from step (c) or(d), optionally under vacuum.

In some embodiments, the formulation of step (a) is filtered prior tostep (b). In some embodiments, the processing step (d) is lyophilizingthe microsphere solution.

In some embodiments, the vessel is a unit dosage container. In someembodiments, the fluid is n-decafluorobutane. In some embodiments, theheadspace of the capped vessel is filled with n-decafluorobutane.

In some embodiments, the mean diameter of the microspheres from step (c)is about 0.1 to 1000 μm. In some embodiments, the mean diameter of themicrospheres from step (c) is about 0.1 to 100 μm. In some embodiments,the mean diameter of the microspheres from step (c) is about 0.1 to 30μm. In some embodiments, the mean diameter of the microspheres from step(c) is about 0.7 to 10 μm.

In some embodiments, the processing step (d) is lyophilizing themicrosphere solution and loss of microspheres during lyophilization isno more than 25%. In some embodiments, the loss of the microspheresduring lyophilization is no more than 15% or no more than 10%.

In some embodiments, the molecular weight of the compound of Formula Iis 1,500 to 5,000 Daltons. In some embodiments, the molecular weight ofthe compound of Formula II is 1,500 to 5,000 Daltons. In someembodiments, the molecular weight of the compound of Formula III is 500to 2,000 Daltons.

In some embodiments, the molar ratio of the compound of Formula I to thecompound of Formula II in the formulation ranges from 1:100 to 1:1. Insome embodiments, the molar ratio of the compound of Formula II to thecompound of Formula III in the formulation ranges from 1:20 to 1:8.

In some embodiments, the microspheres from step (c) comprise 0.01-5 mol% of a compound of Formula I. In some embodiments, the microspheres fromstep (c) comprise 5-9.9 mol % of a compound of Formula II. In someembodiments, the microspheres from step (c) comprise 80-95 mol % of thecompound of Formula III. In some embodiments, the microspheres from step(c) comprise no more than 5 mol % of the compound of Formula I. In someembodiments, the microspheres from step (c) comprise no more than 10 mol% of the compound of Formula II. In some embodiments, the microspheresfrom step (c) comprise no more than 95 mol % of the compound of FormulaIII.

In some embodiments, the compound of Formula I is a compound of FormulaIb; the compound of Formula II is a compound of Formula IIb; and thecompound of Formula III is the compound of Formula IIIa.

In some embodiments, the average molecular weight of the compound ofFormula Ib is about 3200 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula IIb is about 2800 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIIa is about 790 Daltons.

In some embodiments, the microspheres from step (c) comprise 0.01-5 mol% of the compound of Formula Ib. In some embodiments, the microspheresfrom step (c) comprise 5-9.9 mol % of the compound of Formula IIb. Insome embodiments, the microspheres from step (c) comprise 80-95 mol % ofthe compound of Formula IIa.

In some embodiments, the microspheres from step (c) comprise no morethan 5 mol % of the compound of Formula Ib. In some embodiments, themicrospheres from step (c) comprise no more than 10 mol % of thecompound of Formula IIb. In some embodiments, the microspheres from step(c) comprise no more than 95 mol % of the compound of Formula IIIa.

In some embodiments, the molar ratio of the compound of Formula Ib tothe compound of Formula IIb in the microspheres from step (c) rangesfrom 1:100 to 1:1. In some embodiments, the molar ratio of the compoundof Formula IIb to the compound of Formula IIIa in the microspheres fromstep (c) ranges from 1:20 to 1:8.

In yet another aspect, the present disclosure provides a unit dosagecontainer, comprising: a therapeutically effective amount of thepharmaceutical composition provided herein. In some embodiments, thecompound of Formula I is a compound of Formula Ib. In some embodiments,the compound of Formula II is a compound of Formula IIb. In someembodiments, the compound of Formula III is the compound of Formula IIa.In some embodiments, the fluid is air, CO₂, sulfur hexafluoride, afluorinated C₁₋₆ alkane, or a combination thereof. In some embodiments,the fluorinated C₁₋₆ alkane is selected from octafluoropropane,n-decafluorobutane, and dodecafluoropentane. In some embodiments, thefluid is n-decafluorobutane. In some embodiments, the compositioncomprises trehalose and PLASDONE K12 as excipients. In some embodiments,the composition further comprises a buffering solution wherein thebuffering solution is a phosphate salt. In some embodiments, thebuffering solution comprises saline without calcium and magnesium.

In some embodiments, the composition is capable of forming microspheresin the presence of water. In some embodiments, the composition comprisesmicrospheres. In some embodiments, the microspheres exist as lyophilizeddry powder or as water-free concentrate. In some embodiments, thecontainer is prepared by the process provided herein. In someembodiments, the container is sealed by a crimp-top cap fitted with aseptum. In some embodiments, the container is airtight.

In one embodiment, the present disclosure provides a kit comprising: atleast one unit dosage container as described above, and instructions forusing said kit.

In some embodiments, the unit dosage container comprises trehalose andPLASDONE K12 as excipients. In some embodiments, the kit furthercomprises a container comprising an aqueous solution. In someembodiments, the aqueous solution is sterile water. In some embodiments,the aqueous solution is a saline solution ready for perfusion. In someembodiments, the kit further comprises a syringe. In some embodiments,the kit further comprises a needle fitted for the syringe. In someembodiments, the kit comprises a needle-free syringe comprising a sharptip. In some embodiments, the needle or the sharp tip is capable ofpiercing through a septum cap.

In some embodiments, the kit further comprises a container comprising afluid having a normal boiling point of less than 30° C., optionallywherein the container is a syringe. In some embodiments, the fluid isair, nitrogen, argon, CO₂, sulfur hexafluoride, a fluorinated C₁₋₆alkane, or a combination thereof. In some embodiments, the fluorinatedC₁₋₆ alkane is selected from octafluoropropane, n-decafluorobutane, anddodecafluoropentane. In some embodiments, the fluid isn-decafluorobutane.

In some embodiments, the kit further comprises at least one gel pad andultrasound gel. In some embodiments, the kit further comprises anapparatus selected from Mix2Vial® apparatus and a vented vial adapter.

Another aspect of the present disclosure provides a method ofreconstituting microspheres, wherein the method comprises:

-   -   (a) adding a sufficient amount of water or a saline solution to        the microspheres inside the container of the present disclosure,    -   (b) optionally, adding a volume of a fluid having a normal        boiling point of less than 30° C. to the container of step (a);        and    -   (c) optionally shaking the container of step (a) or step (b).

In some embodiments, the method further comprises a step of filling thecontainer with gas before step (a). In some embodiments, the amount ofwater or the saline solution is no more than 100 milliliters. In someembodiments, the amount of the water or the saline solution added issufficient to produce a homogeneous mixture comprising the reconstitutedmicrospheres. In some embodiments, the shaking of the container lasts nomore than 180 seconds.

In yet another aspect, the present disclosure provides a method oftreating urolithiasis, the method comprising: administering to a subjectwith urolithiasis an effective amount of the pharmaceutical composition,microspheres, or the reconstituted microsphere solution disclosed hereinso as to bring the microspheres into contact with the urinary stone, anddirectionally applying an energy, at a frequency that excites the fluidwithin the microspheres, to the urinary stone within the subject.

In some embodiments, the reconstituted microsphere solution isadministered into the ureter of the subject through a urinary catheter.In some embodiments, the energy is in the form of electromagnetic,acoustic, microwave, photonic, or other forms. In some embodiments, theenergy is ultrasonic.

In some embodiments, the ultrasonic energy is in the frequency rangefrom 100 kilohertz (kHz) to 2 megahertz (MHz). In some embodiments, theultrasonic energy is associated with peak pressures in the range 0.1 MPato 10 MPa. In some embodiments, the energy is applied for a sufficientamount of time to fragment the urinary stone.

In some embodiments, the amount of time is no more than 100 minutes. Insome embodiments, the amount of time is no more than 90 minutes. In someembodiments, the amount of time is no more than 80 minutes. In someembodiments, the amount of time is no more than 70 minutes. In someembodiments, the amount of time is no more than 60 minutes. In someembodiments, the amount of time is no more than 50 minutes. In someembodiments, the amount of time is no more than 40 minutes. In someembodiments, the amount of time is no more than 30 minutes. In someembodiments, the amount of time is no more than 25 minutes. In someembodiments, the amount of time is no more than 20 minutes. In someembodiments, the amount of time is no more than 15 minutes. In someembodiments, the amount of time is no more than 10 minutes.

In some embodiments, the applied energy causes a change in volume of thereconstituted microspheres or other cavitation effects of themicrospheres. In some embodiments, the cavitation of the microspherescauses pressure gradient changes and other mechanical effects in theurinary stone around the reconstituted microspheres. In someembodiments, the pressure gradient changes and other mechanical effectsare capable of fragmenting urinary stones. In some embodiments, thesubject is human.

In one aspect, the present disclosure provides a reconstitutedmicrosphere solution for use in the treatment of urolithiasis, whereinan effective amount of the said microsphere solution is administered toa subject so as to bring the microparticles into contact with theurinary stone; and an energy is directionally applied, at a frequencythat excites the fluid within the microsphere, to the urinary stonewithin the subject.

In some embodiments, the reconstituted microsphere solution isadministered into the ureter of the subject through a urinary catheter.In some embodiments, the energy is in the form of electromagnetic,acoustic, microwave, photonic, laser, or other forms. In someembodiments, the energy is ultrasonic.

In some embodiments, the ultrasonic energy is in the frequency rangefrom 100 kilohertz (kHz) to 2 megahertz (MHz). In some embodiments, theultrasonic energy is associated with peak pressures in the range 0.1 MPato 10 MPa.

In some embodiments, the energy is in the form of laser. In someembodiments, the laser energy has a wave length in the infrared rangefrom 1000 nm to 2500 nm. In some embodiments, the laser energy iscapable of vaporizing intraluminal liquid, thereby capable of producingan associated acoustic wave. In some embodiments, the laser energy has afrequency in the range between 1 kHz to 1 MHz.

In some embodiments, the energy is applied for a sufficient amount oftime to fragment the urinary stone. In some embodiments, the amount oftime is no more than 100 minutes. In some embodiments, the amount oftime is no more than 90 minutes. In some embodiments, the amount of timeis no more than 80 minutes. In some embodiments, the amount of time isno more than 70 minutes. In some embodiments, the amount of time is nomore than 60 minutes. In some embodiments, the amount of time is no morethan 50 minutes. In some embodiments, the amount of time is no more than40 minutes. In some embodiments, the amount of time is no more than 30minutes. In some embodiments, the amount of time is no more than 25minutes. In some embodiments, the amount of time is no more than 20minutes. In some embodiments, the amount of time is no more than 15minutes. In some embodiments, the amount of time is no more than 10minutes.

In some embodiments, the applied energy causes a change in volume of thereconstituted microspheres or other cavitation effects of themicrospheres. In some embodiments, the cavitation of the microspherescauses pressure gradient changes and other mechanical effects in theurine around the reconstituted microspheres. In some embodiments, thepressure gradient changes and other mechanical effects are capable offragmenting urinary stones. In some embodiments, the subject is human.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 shows a cutaway schematic of an exemplary microsphere attached toa mineralized material 105, including the accumulation-facilitatingmicrosphere shell constituents 120, the microsphere lipid shell 110, andmicrosphere fluid core 115.

FIG. 2 is a block diagram illustrating exemplary steps of synthesizing abisphosphonate-PEG-lipid provided in this disclosure.

FIG. 3 is a block diagram illustrating exemplary steps of preparing abisphosphonate-PEG-lipid for generating microspheres provided in thisdisclosure.

FIG. 4 shows size distributions for liquid phases of abisphosphonate-PEG-lipid mixture measured by dynamic light scattering(DL S).

FIG. 5 shows samples of a bisphosphonate-PEG-lipid composition with gasheadspace (left) and a microsphere formulation (right) of thecomposition described herein, illustrating qualitative differences ofthe two formulations.

FIG. 6 is a graph showing microsphere accumulation assay results overtime. The graph shows that microspheres incorporatingbisphosphonate-PEG-lipid of the present disclosure have greater rates ofmicrosphere accumulation on a mineralized surface compared tomicrospheres lacking bisphosphonate-PEG-lipid in the shell.

FIG. 7 shows size distribution of microspheres as measured byelectrozone sensing. The number of particles (y-axis) for each bin ofdiameter (x-axis) is provided.

FIG. 8 shows the ¹H-NMR spectrum of intermediate PEG2DA.

FIG. 9 shows the ¹H-NMR spectrum of intermediate1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2)—COOH.

FIG. 10 shows the ³¹P-NMR spectrum of intermediate1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2)—COOH.

FIG. 11 shows the ¹H-NMR spectrum of the compound of Formula Ib.

FIG. 12 shows the ³¹P-NMR spectrum of the compound of Formula Ib.

FIG. 13 shows a gas chromatograph of headspace analysis ofn-decafluorobutane.

FIG. 14 shows the ³¹P-NMR spectrum of the liposome formulationcomprising the compound of Formula Ib,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) (DSPE-PEG2K, a compound of Formula IIb)and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, a compound ofFormula IIIa).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise defined herein, all technical and scientific terms usedherein have the meaning commonly understood by a person skilled in theart to which this invention pertains.

The term “subject” refers to any mammal including humans, and mammalssuch as those animals of veterinary and research interest that areincluding, but not limited to: simians, cattle, horses, dogs, cats, androdents.

The term “treating” or “treatment of” a disorder or disease refers totaking steps to alleviate the symptoms of the disorder or disease, e.g.,reduction, destruction or removal of an abnormal or obstructive masssuch as kidney stones, urinary stones, biliary stones, blood clots,fibroids, cancerous tumors, and atheromatous plaques, or otherwiseobtain some beneficial or desired results for a subject, includingclinical results. Any beneficial or desired clinical results mayinclude, but are not limited to, alleviation or amelioration of one ormore symptoms of the disorder or disease; diminishment of the extent ofthe disease; delay or slowing disease progression; amelioration,palliation, or stabilization of the disease state; or other beneficialresults.

The term “effective amount” means an amount sufficient to produce adesired effect.

The term “sufficient amount” means an amount sufficient to produce adesired effect.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate or reduce a symptom of a disease.

The term “reduction” of a symptom or symptoms (and grammaticalequivalents of this phrase) refers to decreasing the severity orfrequency of the symptom(s), or elimination of the symptom(s).

The term “cavitational effect” as used herein refers to an effectsufficient to cause reduction or destruction of an abnormal orobstructive mass in vivo such as kidney stones, urinary stones, biliarystones, blood clots, fibroids, cancerous tumors, and atheromatousplaques.

The term “PL2kS” as used herein refers to the compound1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000-alendronate] (ammonium salt), which is also disclosed asthe compound of Formula Ib herein.

The term “DSPE-PEG2K” as used herein refers to the compound1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt), which is also disclosed as the compoundof Formula IIb herein.

The term “DSPC” as used herein refers to the compound1,2-distearoyl-sn-glycero-3-phosphocholine, which is also disclosed asthe compound of Formula IIIa herein.

The practice of the present invention includes the use of conventionaltechniques of organic chemistry, molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art.

In this application, reference will be made to a number of technicaldesignations. All numerical designations, e.g., pH, temperature, time,concentration, and weight, including ranges of each thereof, areapproximations that typically may be varied (+) or (−) by increments of0.1, 1.0, or 10.0, as appropriate. All numerical designations may beunderstood as preceded by the term “about.” Reagents described hereinare exemplary and equivalents of such may be known in the art.

Compounds utilized in the present invention may possess asymmetriccarbon atoms (optical centers) or double bonds; the racemates,diastereomers, geometric isomers, regioisomers and individual isomers(e.g., separate enantiomers) are all intended to be encompassed withinthe scope of the present invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example, and withoutlimitation, tritium (³H), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). Allisotopic variations of the compounds of the present invention, whetherradioactive or not, are intended to be encompassed within the scope ofthe present invention.

Stereoisomers of compounds (also known as optical isomers) include allchiral, diastereomeric, and racemic forms of a structure, unless thespecific stereochemistry is expressly indicated. Thus, compounds used inthe present technology include enriched or resolved optical isomers atany or all asymmetric atoms as are apparent from the depictions. Bothracemic and diastereomeric mixtures, as well as the individual opticalisomers can be isolated or synthesized so as to be substantially free oftheir enantiomeric or diastereomeric partners, and these stereoisomersare all within the scope of the present technology.

The compounds of the present technology can exist as solvates,especially hydrates. Hydrates may form during manufacture of thecompounds or compositions comprising the compounds, or hydrates may formover time due to the hygroscopic nature of the compounds. Compounds ofthe present technology can exist as organic solvates as well, includingDMF, ether, and alcohol solvates among others. The identification andpreparation of any particular solvate is within the skill of theordinary artisan of synthetic organic or medicinal chemistry.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment.

Stereoisomers of compounds (also known as optical isomers) include allchiral, diastereomeric, and racemic forms of a structure, unless thespecific stereochemistry is expressly indicated. Thus, compounds used inthe present technology include enriched or resolved optical isomers atany or all asymmetric atoms as are apparent from the depictions. Bothracemic and d or 1 enriched stereomeric mixtures, as well as theindividual optical isomers can be isolated or synthesized so as to besubstantially free of their enantiomeric or diastereomeric partners, andthese stereoisomers are all within the scope of the present technology.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,etc.) are not intended for inclusion herein. In such cases, the maximumnumber of such substituents is three. That is to say that each of theabove definitions is constrained by a limitation that each functionalgroup is substituted (at from one to three positions) and that any andall of those substituent groups may be substituted one more time (atfrom one to three positions).

It is understood that the above definitions are not intended to includeimpermissible substitution patterns (e.g., methyl substituted with 5fluoro groups). Such impermissible substitution patterns are well knownto the skilled artisan.

Throughout this application, the text refers to various embodiments ofthe present compounds, compositions, and methods. The variousembodiments described are meant to provide a variety of illustrativeexamples and should not be construed as descriptions of alternativespecies. Rather, it should be noted that the descriptions of variousembodiments provided herein may be of overlapping scope. The embodimentsdiscussed herein are merely illustrative and are not meant to limit thescope of the present technology.

Compounds

The present disclosure provides compounds that exhibit bothhydrophobicity and a high affinity for calcium and other metals inmineralized forms, including biominerals. The compounds can be used in avariety of applications in which both hydrophobicity and binding tometal-containing materials are desired.

More specifically, in a first aspect, the present disclosure provides acompound of Formula IV

or a salt, isomer, or salt of an isomer thereof, wherein:

p is from 10 to 30;

q is from 1 to 100;

C is selected from the group consisting of:

B is selected from the group consisting of: a covalent bond and an ethylgroup;

A is selected from the group consisting of: a covalent bond, acyl,acylamino, aminoacyl, acyloxy, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyloxy,aminosulfonylamino, aminosulfonyl, amidino, and carboxy ester; and

X is selected from the group consisting of: hydrogen, silyl, acyl,aminoacyl, thioacyl, aminocarbonyl, aminoacyl carbonyloxy,aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl,substituted sulfinyl, carboxy ester, phthalimido, OH, SO₃H and PO₃H.

In some embodiments, p is from 12 to 28, from 14 to 26, from 14 to 24,from 14 to 22, from 14 to 20, from 16 to 20, or from 16 to 18. In someembodiments, p is 16. In some embodiments, q is from 1 to 100, from 5 to90, from 10 to 80, from 15 to 70, from 20 to 60, from 25 to 50, from 30to 50, from 40 to 50, or from 40 to 45. In some embodiments, q is 43. Insome embodiments, q is 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, or50. In some embodiments, C is

In some embodiments, the compound of Formula IV is a compound of FormulaI

or a salt, isomer, or salt of an isomer thereof, wherein:

n is from 10 to 30,

m is from 1 to 100.

In some embodiments, n is from 20 to 25, from 12 to 20, from 14 to 18,or from 14 to 16. In some embodiments, n is 14 or 16. In someembodiments, m is from 1 to 90, from 5 to 80, from 10 to 70, from 20 to60, from 30 to 50, or from 38 to 50. In some embodiments, m is 43. Insome embodiments, q is 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, or50. In some embodiments, X is selected from the group consisting of:hydrogen, silyl, acyl, aminoacyl, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminosulfonyl, amidino, substitutedsulfonyl, substituted sulfinyl, carboxy ester, phthalimido, OH, 503H andPO₃H.

In some embodiments, the compound of Formula I is a compound of FormulaIa

or a salt, isomer, or salt of an isomer thereof.

In some embodiments, the compound of Formula I is a compound of FormulaIb

Pharmaceutical Compositions

In a second aspect, the present disclosure provides for a pharmaceuticalcomposition comprising

a compound of Formula IV

or a pharmaceutically acceptable salt, isomer, or salt of an isomerthereof, wherein:

p is from 10 to 30;

q is from 1 to 100;

C is selected from the group consisting of:

B is selected from the group consisting of: a covalent bond and an ethylgroup;

A is selected from the group consisting of: a covalent bond, acyl,acylamino, aminoacyl, acyloxy, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyloxy,aminosulfonylamino, aminosulfonyl, amidino, and carboxy ester; and

X is selected from the group consisting of: hydrogen, silyl, acyl,aminoacyl, thioacyl, aminocarbonyl, aminoacyl carbonyloxy,aminothiocarbonyl, aminosulfonyl, amidino, substituted sulfonyl,substituted sulfinyl, carboxy ester, phthalimido, SO₃H and PO₃H; andoptionally, a pharmaceutically acceptable excipient or diluent

In some embodiments, p is from 12 to 28, from 14 to 26, from 14 to 24,from 14 to 22, from 14 to 20, from 16 to 20, or from 16 to 18. In someembodiments, p in the pharmaceutical composition is 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20. In some embodiments, an average value of p inthe pharmaceutical composition is from 12 to 28, from 14 to 26, from 14to 24, from 14 to 22, from 14 to 20, from 16 to 20, or from 16 to 18. Insome embodiments, an average value of p in the pharmaceuticalcomposition is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

In some embodiments, q is from 1 to 100, from 5 to 90, from 10 to 80,from 15 to 70, from 20 to 60, from 25 to 50, from 30 to 50, from 40 to50, or from 40 to 45. In some embodiments, q is 43. In some embodiments,q is 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, or 50. In someembodiments, an average value of q in the pharmaceutical composition isfrom 1 to 100, from 5 to 90, from 10 to 80, from 15 to 70, from 20 to60, from 25 to 50, from 30 to 50, from 40 to 50, or from 40 to 45. Insome embodiments, an average value of q in the pharmaceuticalcomposition is 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, or 50.

In some embodiments of the pharmaceutical composition, molecular weightof the compound of Formula IV is 1,500 to 5,000 Daltons. In certainembodiments, the molecular weight of the compound of Formula IV is 1,500to 5,000 Daltons. In certain embodiments, the molecular weight of thecompound of Formula IV is 2,000 to 4,500 Daltons. In certainembodiments, the molecular weight of the compound of Formula IV is 2,500to 4,000 Daltons. In certain embodiments, the molecular weight of thecompound of Formula IV is 3,000 to 3,500 Daltons

In some embodiments, C is

In some embodiments of the pharmaceutical composition, the compound ofFormula IV is a compound of Formula I

or a salt, isomer, or salt of an isomer thereof, wherein:

n is from 10 to 30 and

m is from 1 to 100.

In some embodiments, n is from 10 to 50, from 20 to 25, from 12 to 20,from 14 to 18, or from 14 to 16. In some embodiments, n is 14 or 16. Insome embodiments, n is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Insome embodiments, an average value of n in the pharmaceuticalcomposition is from 10 to 50, from 20 to 25, from 12 to 20, from 14 to18, or from 14 to 16. In some embodiments, an average value of n in thepharmaceutical composition is 14 or 16. In some embodiments, an averagevalue of n in the pharmaceutical composition is 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20.

In some embodiments, m is from 1 to 150, from 1 to 140, from 1 to 130,from 1 to 120, 1 to 110, from 1 to 100, from 1 to 90, from 5 to 80, from10 to 70, from 20 to 60, from 30 to 50, or from 38 to 50. In someembodiments, m is 43. In some embodiments, m is 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 49, or 50. In some embodiments, an average value of m inthe pharmaceutical composition is from 1 to 150, from 1 to 140, from 1to 130, from 1 to 120, 1 to 110, from 1 to 100, from 1 to 90, from 5 to80, from 10 to 70, from 20 to 60, from 30 to 50, or from 38 to 50. Insome embodiments, an average value of m in the pharmaceuticalcomposition is 43. In some embodiments, an average value of m in thepharmaceutical composition is 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,49, or 50.

In some embodiments of the pharmaceutical composition, the molecularweight of the compound of Formula I is 1,500 to 5,000 Daltons. Incertain embodiments, the molecular weight of the compound of Formula Iis 2,000 to 4,500 Daltons. In certain embodiments, the molecular weightof the compound of Formula I is 2,500 to 4,000 Daltons. In certainembodiments, the molecular weight of the compound of Formula I is 3,000to 3,500 Daltons.

In some embodiments, X is selected from the group consisting of:hydrogen, silyl, acyl, aminoacyl, thioacyl, aminocarbonyl, aminoacylcarbonyloxy, aminothiocarbonyl, aminosulfonyl, amidino, substitutedsulfonyl, substituted sulfinyl, carboxy ester, phthalimido, OH, SO₃H andPO₃H.

In some embodiments, the compound of Formula I is a compound of FormulaIa

or a salt, isomer or salt of an isomer thereof.

In some embodiments of the pharmaceutical composition, the averagemolecular weight of the compound of Formula Ia is about 3500 Daltons. Insome embodiments, the average molecular weight of the compound ofFormula Ia is about 3200 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula Ia is about 2500 Daltons. Insome embodiments, the average molecular weight of the compound ofFormula Ia is about 2000 Daltons.

In some embodiments of the pharmaceutical composition, the compound ofFormula Ia is the compound of Formula Ib

In some embodiments, the compound of Formula I is the compound ofFormula Ib

In some embodiments of the pharmaceutical composition, the averagemolecular weight of the compound of Formula Ib is about 3500 Daltons. Insome embodiments, the average molecular weight of the compound ofFormula Ib is about 3200 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula Ib is about 2500 Daltons. Insome embodiments, the average molecular weight of the compound ofFormula Ib is about 2000 Daltons.

In some embodiments, chain lengths (16 and 43) in Formula Ib is anaverage chain length of the compound in the pharmaceutical composition.

In some embodiments of the pharmaceutical composition, the compositionfurther comprises at least one additional phospholipid compound.

In some embodiments, the pharmaceutical composition further comprises

a compound of Formula II

or a pharmaceutically acceptable salt or isomer thereof.

In some embodiments, t is from 10 to 30, from 12 to 28, from 14 to 25,from 16 to 22, from 16 to 20, or from 18 to 20. In some embodiments, tis 14 or 16. In some embodiments, an average value oft in thepharmaceutical composition is from 10 to 30, from 12 to 28, from 14 to25, from 16 to 22, from 16 to 20, or from 18 to 20. In some embodiments,an average value of t in the pharmaceutical composition is 14 or 16.

In some embodiments, y is from 1-100, from 5 to 90, from 10 to 80, from20 to 70, from 40 to 60, or from 40 to 50. In some embodiments, y isfrom 38 to 50. In some embodiments, an average value of y in thepharmaceutical composition is from 1-100, from 5 to 90, from 10 to 80,from 20 to 70, from 40 to 60, or from 40 to 50. In some embodiments, anaverage value of y in the pharmaceutical composition is from 38 to 50.

In some embodiments of the pharmaceutical composition, the molecularweight of the compound of Formula II is 1,500 to 5,000 Daltons. Incertain embodiments, the molecular weight of the compound of Formula IIis 2,000 to 4,500 Daltons. In certain embodiments, the molecular weightof the compound of Formula II is 2,500 to 4,000 Daltons. In particularembodiments, the molecular weight of the compound of Formula II is 3,000to 3,500 Daltons.

In some embodiments of the pharmaceutical composition, the compound ofFormula II is a compound of Formula IIa

or a pharmaceutically acceptable salt or isomer thereof.

In some embodiments of the pharmaceutical composition, the averagemolecular weight of the compound of Formula IIa is about 3000 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIa is about 2800 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula IIa is about 2600 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIa is about 2400 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula IIa is about 2200 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIa is about 2000 Daltons.

In some embodiments, chain lengths (16 and 43) in Formula IIa is anaverage chain length of the compound in the pharmaceutical composition.

In some embodiments of the pharmaceutical composition, the compound ofFormula II is the compound of Formula IIb)

In some embodiments of the pharmaceutical composition, the averagemolecular weight of the compound of Formula IIb is about 3000 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIb is about 2800 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula IIb is about 2600 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIb is about 2400 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula IIb is about 2200 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIb is about 2000 Daltons.

In some embodiments, chain lengths (16 and 43) in Formula IIb is anaverage chain length of the compound in the pharmaceutical composition.

In some embodiments, the pharmaceutical composition further comprises

a compound of Formula III

or a pharmaceutically acceptable salt or isomer thereof. In someembodiments, z is from 10 to 30, from 10 to 25, from 12 to 20, from 15to 20, or from 16 to 18. In some embodiments, z is 14 or 16. In someembodiments, an average value of z in the pharmaceutical composition isfrom 10 to 30, from 10 to 25, from 12 to 20, from 15 to 20, or from 16to 18. In some embodiments, an average value of z in the pharmaceuticalcomposition is 14 or 16.

In some embodiments of the pharmaceutical composition, the molecularweight of the compound of Formula III is 500 to 2,000 Daltons. Incertain embodiments, the molecular weight of the compound of Formula IIIis 750 to 1,750 Daltons. In certain embodiments, the molecular weight ofthe compound of Formula III is 1,000 to 1,500 Daltons. In certainembodiments, the molecular weight of the compound of Formula III is1,100 to 1,400 Daltons. In certain embodiments, the molecular weight ofthe compound of Formula III is 1200 to 1,300 Daltons.

In some embodiments of the pharmaceutical composition, the compound ofFormula III is the compound of Formula IIIa

In some embodiments of the pharmaceutical composition, the averagemolecular weight of the compound of Formula IIIa is about 840 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIIa is about 790 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula IIIa is about 750 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIIa is about 700 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula IIIa is about 650 Daltons.

In some embodiments, the chain length (16) in Formula IIIa is an averagechain length of the compound in the pharmaceutical composition.

In some embodiments, the pharmaceutical composition comprises no morethan 5 mol % of the compound of Formula IV or a salt thereof. Mol % asused herein refers to a percentage of the number of molecules of a givenlipid compound (e.g., the compound of Formula IV, Formula I, Formula II,Formula III, or a salt, isomer, or salt of an isomer thereof) over totalnumber of molecules of all lipid compounds within the pharmaceuticalcomposition. Similarly, in the context of microspheres, mol % as usedherein refers to a percentage of the number of molecules of a givenlipid compound (e.g., the compound of Formula IV, Formula I, Formula II,Formula III, or a salt, isomer, salt of an isomer thereof) over totalnumber of molecules of all lipid compounds within the microspheres. Incertain embodiments, the pharmaceutical composition comprises no morethan 4 mol % of the compound of Formula IV or a salt thereof. In certainembodiments, the pharmaceutical composition comprises no more than 4 mol% of the compound of Formula IV or a salt thereof. In certainembodiments, the pharmaceutical composition comprises no more than 3 mol% of the compound of Formula IV or a salt thereof. In certainembodiments, the pharmaceutical composition comprises no more than 2 mol% of the compound of Formula IV or a salt thereof. In certainembodiments, the pharmaceutical composition comprises no more than 1 mol% of the compound of Formula IV or a salt thereof.

In some embodiments, the pharmaceutical composition comprises 0.1-5 mol% of the compound of Formula IV or a salt thereof. In some embodiments,the pharmaceutical composition comprises 0.1-10, 0.1-5, 0.5-5, 1-5, 1-4,1-3, 2-3, 2-4, or 3-5 mol % of the compound of Formula IV or a saltthereof.

In some embodiments, the composition comprises 0.01-5 mol % of thecompound of Formula I, or a salt thereof. In certain embodiments, thecomposition comprises 0.1-5 mol % of the compound of Formula I, or asalt thereof. In some embodiments, the pharmaceutical compositioncomprises 0.1-10, 0.1-5, 0.5-5, 1-5, 1-4, 1-3, 2-3, 2-4, or 3-5 mol % ofthe compound of Formula I or a salt thereof.

In some embodiments, the pharmaceutical composition comprises no morethan 5 mol % of the compound of Formula I or a salt thereof. In certainembodiments, the pharmaceutical composition comprises no more than 4 mol% of the compound of Formula I or a salt thereof. In certainembodiments, the pharmaceutical composition comprises no more than 3 mol% of the compound of Formula I or a salt thereof. In certainembodiments, the pharmaceutical composition comprises no more than 2 mol% of the compound of Formula I or a salt thereof. In certainembodiments, the pharmaceutical composition comprises no more than 1 mol% of the compound of Formula I or a salt thereof.

In some embodiments, the pharmaceutical composition comprises 0.01-5 mol% of a compound of Formula Ia or Ib. In certain embodiments, thepharmaceutical composition comprises 0.1-5 mol % of a compound ofFormula Ia or Ib. In certain embodiments, the composition comprises0.5-4.5 mol % of a compound of Formula Ia or Ib. In certain embodiments,the composition comprises 1.0-4.0 mol % of a compound of Formula Ia orIb. In certain embodiments, the composition comprises 1.5-3.5 mol % of acompound of Formula Ia or Ib. In certain embodiments, the compositioncomprises 2.0-3.0 mol % of a compound of Formula Ia or Ib.

In some embodiments, the composition comprises no more than 5 mol % of acompound of Formula Ia or Ib. In some embodiments, the compositioncomprises no more than 4.5 mol % of a compound of Formula Ia or Ib. Insome embodiments, the composition comprises no more than 4.0 mol % of acompound of Formula Ia or Ib. In some embodiments, the compositioncomprises no more than 3.5 mol % of a compound of Formula Ia or Ib. Insome embodiments, the composition comprises no more than 3.0 mol % of acompound of Formula Ia or Ib. In some embodiments, the compositioncomprises no more than 2.5 mol % of a compound of Formula Ia or Ib. Insome embodiments, the composition comprises no more than 2.0 mol % of acompound of Formula Ia or Ib. In some embodiments, the compositioncomprises no more than 1.5 mol % of a compound of Formula Ia or Ib. Insome embodiments, the composition comprises no more than 1.0 mol % of acompound of Formula Ia or Ib. In some embodiments, the compositioncomprises no more than 0.5 mol % of a compound of Formula Ia or Ib. Insome embodiments, the composition comprises no more than 0.1 mol % of acompound of Formula Ia or Ib. In some embodiments, the compositioncomprises no more than 0.05 mol % of a compound of Formula Ia or Ib.

In some embodiments, the composition comprises 5-10 mol % of thecompound of Formula II, or a salt thereof.

In some embodiments, the pharmaceutical composition comprises no morethan 10 mol % of the compound of Formula II or a salt thereof. Incertain embodiments, the pharmaceutical composition comprises no morethan 8 mol % of the compound of Formula II or a salt thereof. In certainembodiments, the composition comprises no more than 6 mol % of thecompound of Formula II or a salt thereof. In certain embodiments, thepharmaceutical composition comprises no more than 4 mol % of thecompound of Formula II or a salt thereof. In certain embodiments, thepharmaceutical composition comprises no more than 3 mol % of thecompound of Formula II or a salt thereof. In certain embodiments, thepharmaceutical composition comprises no more than 2 mol % of thecompound of Formula II or a salt thereof. In certain embodiments, thepharmaceutical composition comprises no more than 1 mol % of thecompound of Formula II or a salt thereof.

In some embodiments, the pharmaceutical composition comprises 5-10 mol %of the compound of Formula II or a salt thereof. In some embodiments,the pharmaceutical composition comprises 5-20, 5-15, 5-10, 5-9.9, 5-9.6,5-9, 5-9, 7-9, or 8-9 mol % of the compound of Formula II or a saltthereof.

In some embodiments, the composition comprises 5.0-10.0 mol % of acompound of Formula IIa or IIb. In certain embodiments, the compositioncomprises 5.5-9.5 mol % of a compound of Formula IIa or IIb. In certainembodiments, the composition comprises 6.0-9.0 mol % of a compound ofFormula IIa or IIb. In certain embodiments, the composition comprises6.5-8.5 mol % of a compound of Formula IIa or IIb. In certainembodiments, the composition comprises 7.0-8.0 mol % of a compound ofFormula IIa or IIb.

In some embodiments, the composition comprises no more than 10 mol % ofa compound of Formula IIa or IIb. In some embodiments, the compositioncomprises no more than 9.5 mol % of a compound of Formula IIa or IIb. Insome embodiments, the composition comprises no more than 9.0 mol % of acompound of Formula IIa or IIb. In some embodiments, the compositioncomprises no more than 8.5 mol % of a compound of Formula IIa or IIb. Insome embodiments, the composition comprises no more than 8.0 mol % of acompound of Formula IIa or IIb. In some embodiments, the compositioncomprises no more than 7.5 mol % of a compound of Formula IIa or IIb. Insome embodiments, the composition comprises no more than 7.0 mol % of acompound of Formula IIa or IIb. In some embodiments, the compositioncomprises no more than 6.5 mol % of a compound of Formula IIa or IIb. Insome embodiments, the composition comprises no more than 6.0 mol % of acompound of Formula IIa or IIb. In some embodiments, the compositioncomprises no more than 5.5 mol % of a compound of Formula IIa or IIb. Insome embodiments, the composition comprises no more than 5.0 mol % of acompound of Formula IIa or IIb.

In some embodiments, the composition comprises 80-95 mol % of thecompound of Formula III, or a salt thereof. In some embodiments, thepharmaceutical composition comprises 70-99, 75-98, 75-97, 80-95 mol % ofthe compound of Formula III or a salt thereof.

In some embodiments, the pharmaceutical composition comprises no morethan 95 mol % of the compound of Formula III or a salt thereof. Incertain embodiments, the pharmaceutical composition comprises no morethan 85 mol % of the compound of Formula III or a salt thereof. Incertain embodiments, the pharmaceutical composition comprises no morethan 75 mol % of the compound of Formula III or a salt thereof. Incertain embodiments, the pharmaceutical composition comprises no morethan 65 mol % of the compound of Formula III or a salt thereof. Incertain embodiments, the pharmaceutical composition comprises no morethan 55 mol % of the compound of Formula III or a salt thereof. Incertain embodiments, the pharmaceutical composition comprises no morethan 45 mol % of the compound of Formula III or a salt thereof. Incertain embodiments, the pharmaceutical composition comprises no morethan 35 mol % of the compound of Formula III or a salt thereof. Incertain embodiments, the pharmaceutical composition comprises no morethan 25 mol % of the compound of Formula III or a salt thereof. Incertain embodiments, the pharmaceutical composition comprises no morethan 15 mol % of the compound of Formula III or a salt thereof. Incertain embodiments, the pharmaceutical composition comprises no morethan 5 mol % of the compound of Formula III or a salt thereof.

In some embodiments, the composition comprises 80-95 mol % of thecompound of Formula IIIa. In certain embodiments, the compositioncomprises 82.5-92.5 mol % of the compound of Formula IIIa. In certainembodiments, the composition comprises 85-90 mol % of the compound ofFormula IIIa. In certain embodiments, the composition comprises87.5-92.5 mol % of the compound of Formula IIIa.

In some embodiments of the pharmaceutical composition, the molar ratioof the compound of Formula IV to the compound of Formula II ranges from1:1000 to 1:1. In certain embodiments of the pharmaceutical composition,the molar ratio of the compound of Formula IV to the compound of FormulaII ranges from 1:500 to 1:1. In certain embodiments of thepharmaceutical composition, the molar ratio of the compound of FormulaIV to the compound of Formula II ranges from 1:100 to 1:1. In certainembodiments, the molar ratio of the compound of Formula IV to thecompound of Formula II ranges from 1:90 to 1:5. In certain embodiments,the molar ratio of the compound of Formula IV to the compound of FormulaII ranges from 1:80 to 1:10. In certain embodiments, the molar ratio ofthe compound of Formula IV to the compound of Formula II ranges from1:70 to 1:20. In certain embodiments, the molar ratio of the compound ofFormula IV to the compound of Formula II ranges from 1:60 to 1:30. Incertain embodiments, the molar ratio of the compound of Formula IV tothe compound of Formula II ranges from 1:50 to 1:40.

In some embodiments of the pharmaceutical composition, the molar ratioof the compound of Formula I to the compound of Formula II ranges from1:1000 to 1:1. In certain embodiments of the pharmaceutical composition,the molar ratio of the compound of Formula I to the compound of FormulaII ranges from 1:500 to 1:1. In certain embodiments of thepharmaceutical composition, the molar ratio of the compound of Formula Ito the compound of Formula II ranges from 1:100 to 1:1. In certainembodiments, the molar ratio of the compound of Formula I to thecompound of Formula II ranges from 1:90 to 1:5. In certain embodiments,the molar ratio of the compound of Formula I to the compound of FormulaII ranges from 1:80 to 1:10. In certain embodiments, the molar ratio ofthe compound of Formula I to the compound of Formula II ranges from 1:70to 1:20. In certain embodiments, the molar ratio of the compound ofFormula I to the compound of Formula II ranges from 1:60 to 1:30. Incertain embodiments, the molar ratio of the compound of Formula I to thecompound of Formula II ranges from 1:50 to 1:40.

In some embodiments of the pharmaceutical composition, the molar ratioof the compound of Formula Ia to the compound of Formula IIa ranges from1:1000 to 1:1. In some embodiments of the pharmaceutical composition,the molar ratio of the compound of Formula Ia to the compound of FormulaIIa ranges from 1:500 to 1:1. In some embodiments of the pharmaceuticalcomposition, the molar ratio of the compound of Formula Ia to thecompound of Formula IIa ranges from 1:100 to 1:1. In some embodiments,the molar ratio of the compound of Formula Ia to the compound of FormulaIIa ranges from 1:90 to 1:5. In some embodiments, the molar ratio of thecompound of Formula Ia to the compound of Formula IIa ranges from 1:80to 1:10. In some embodiments, the molar ratio of the compound of FormulaIa to the compound of Formula IIa ranges from 1:70 to 1:20. In someembodiments, the molar ratio of the compound of Formula Ia to thecompound of Formula IIa ranges from 1:60 to 1:30. In some embodiments,the molar ratio of the compound of Formula Ia to the compound of FormulaIIa ranges from 1:50 to 1:40.

In some embodiments of the pharmaceutical composition, the molar ratioof the compound of Formula Ib to the compound of Formula III) rangesfrom 1:1000 to 1:1. In some embodiments of the pharmaceuticalcomposition, the molar ratio of the compound of Formula Ib to thecompound of Formula IIb ranges from 1:500 to 1:1. In some embodiments ofthe pharmaceutical composition, the molar ratio of the compound ofFormula Ib to the compound of Formula IIb ranges from 1:100 to 1:1. Insome embodiments, the molar ratio of the compound of Formula Ib to thecompound of Formula IIb ranges from 1:90 to 1:5. In some embodiments,the molar ratio of the compound of Formula Ib to the compound of FormulaIIb ranges from 1:80 to 1:10. In some embodiments, the molar ratio ofthe compound of Formula Ib to the compound of Formula IIb ranges from1:70 to 1:20. In some embodiments, the molar ratio of the compound ofFormula Ib to the compound of Formula IIb ranges from 1:60 to 1:30. Insome embodiments, the molar ratio of the compound of Formula Ib to thecompound of Formula IIb ranges from 1:50 to 1:40.

In some embodiments of the pharmaceutical composition, the molar ratioof the compound of Formula II to the compound of Formula III ranges from1:20 to 1:8. In certain embodiments, the molar ratio of the compound ofFormula II to the compound of Formula III ranges from 1:18 to 1:10. Incertain embodiments, the molar ratio of the compound of Formula II tothe compound of Formula III ranges from 1:16 to 1:12. In certainembodiments, the molar ratio of the compound of Formula II to thecompound of Formula III ranges from 1:15 to 1:13.

In some embodiments of the pharmaceutical composition, the molar ratioof the compound of Formula IIa to the compound of Formula IIIa rangesfrom 1:20 to 1:8. In some embodiments, the molar ratio of the compoundof Formula IIa to the compound of Formula IIIa ranges from 1:18 to 1:10.In some embodiments, the molar ratio of the compound of Formula IIa tothe compound of Formula IIIa ranges from 1:16 to 1:12. In someembodiments, the molar ratio of the compound of Formula IIa to thecompound of Formula IIIa ranges from 1:15 to 1:13.

In some embodiments of the pharmaceutical composition, the molar ratioof the compound of Formula IIb to the compound of Formula IIIa rangesfrom 1:20 to 1:8. In some embodiments, the molar ratio of the compoundof Formula IIb to the compound of Formula IIIa ranges from 1:18 to 1:10.In some embodiments, the molar ratio of the compound of Formula IIb tothe compound of Formula IIIa ranges from 1:16 to 1:12. In someembodiments, the molar ratio of the compound of Formula IIb to thecompound of Formula IIIa ranges from 1:15 to 1:13.

In some embodiments, the pharmaceutical composition comprises a compoundof Formula IV, optionally as Formula I, or a pharmaceutically acceptablesalt, isomer, or salt of an isomer thereof a compound of Formula II or apharmaceutically acceptable salt, isomer, or salt of an isomer thereof;and a compound of Formula III or a pharmaceutically acceptable salt,isomer, or salt of an isomer thereof.

In some embodiments, the pharmaceutical composition comprises theammonium salt of at least one of the compounds of Formula IV, Formula I,Formula II, and Formula III.

In some embodiments, the pharmaceutical composition comprises ahomogenous population of a compound having identical carbon chainlengths. In some embodiments, the pharmaceutical composition comprises aheterogenous population of a lipid compound having various lengths foreach carbon chain. In such embodiments, chain lengths represented as avariable or a specific number in Formula IV, Formula I, Formula Ia,Formula Ib, Formula II, Formula IIa, Formula IIb, Formula III, orFormula IIIa refer to an average number of chain links of the lipidcomposition present in the pharmaceutical composition.

In some embodiments, the composition further comprises a fluid with anormal boiling point less than 30° C.

In typical embodiments, the fluid is a gas at the temperatures andpressures present in the mammalian urinary tract. In preferredembodiments, the fluid is responsive to energy of various forms, such aselectromagnetic energy, acoustic energy, microwave energy, photonicenergy, or others. In certain embodiments, the fluid has low solubilityin aqueous solutions, and is air, nitrogen, argon, CO₂, sulfurhexafluoride, a fluorinated C₁₋₆ alkane, or a combination thereof.

In some embodiments of the pharmaceutical composition, the fluid is aperfluorocarbon. In some embodiments, the fluid is selected fromoctafluoropropane, n-decafluorobutane, n-perfluoropropane,tetradecafluorohexane, and dodecafluoropentane. In a particularembodiment, the fluid is n-decafluorobtuane (a.k.a., perfluorobutane).

In some embodiments of the pharmaceutical composition, the compound offormula IV, which in typical embodiments is a compound of formula I; thecompound of formula II; the compound of formula III; and the liquid witha normal boiling point less than 30° C. are capable of assembling intomicrospheres. Thus, in some embodiments, the pharmaceutical compositioncomprises microspheres, the microspheres comprising a lipid shellsurrounding a fluid with normal boiling point less than 30° C., whereinthe lipid shell comprises compounds of formulae I, II and III.

A mean diameter of the microspheres can be measured using any of themethods known in the art. In some embodiments, the mean diameter ismeasured by electro-zone sensing, for example using a Coulter counter.In some embodiments, the microspheres have a mean diameter of about 0.1micron to about 10 microns, or 0.1 microns to 10 microns when measuredby electro-zone sensing. In certain embodiments, the microspheres have amean diameter of about 0.2 micron to about 9.5 microns. In certainembodiments, the microspheres have a mean diameter of about 0.3 micronto about 9.0 microns. In certain embodiments, the microspheres have amean diameter of about 0.5 micron to about 8.5 microns. In certainembodiments, the microspheres have a mean diameter of about 0.5 micronto about 8.0 microns. In certain embodiments, the microspheres have amean diameter of about 0.5 micron to about 7.5 microns. In certainembodiments, the microspheres have a mean diameter of about 0.5 micronto about 7.0 microns. In certain embodiments, the microspheres have amean diameter of about 0.5 micron to about 6.5 microns. In certainembodiments, the microspheres have a mean diameter of about 0.5 micronto about 6.0 microns. In certain embodiments, the microspheres have amean diameter of about 0.5 micron to about 5.5 microns. In certainembodiments, the microspheres have a mean diameter of about 0.5 micronto about 5 microns. In certain embodiments, the microspheres have a meandiameter of about 0.5 micron to about 4 microns. In certain embodiments,the microspheres have a mean diameter of about 0.5 micron to about 3microns. In certain embodiments, the microspheres have a mean diameterof about 0.5 micron to about 2 microns. In certain embodiments, themicrospheres have a mean diameter of about 0.5 micron, 0.6 micron, 0.7micron, 0.8 micron, 0.9 micron, 1 micron, or 5 microns.

In some embodiments, the pharmaceutical composition comprisingmicrospheres is a lyophilized powder or dry concentrate.

In other embodiments, the pharmaceutical composition comprisingmicrospheres further comprises an aqueous medium, such as water, saline,or a buffered salt solution. In certain embodiments, the pharmaceuticalcomposition comprises a phosphate buffered solution lacking calcium andmagnesium ions. In particular embodiments, the composition furthercomprises phosphate buffered saline.

In some embodiments, the pharmaceutical composition comprisingmicrospheres, whether lyophilized, dry concentrate, or liquidformulation, further comprises one or more of trehalose and PLASDONE K12as excipients. PLASDONE K12 is a commercially available and product.

Method of Manufacturing Bisphosphonate-PEG-Lipid Compounds

In another aspect, the present disclosure provides methods of makingbisphosphonate-PEG-lipid compounds.

An exemplary method of making bisphosphonate-PEG-lipid compounds isillustrated in FIG. 2. The method comprises the steps of (i)synthesizing dioic acid form of polyethylene glycol compounds byoxoammonium-catalyzed oxidation; (ii) synthesizing PEG-lipid by NHSesterification and chromatography; and (iii) synthesizingbisphosphonate-PEG-lipid by NHS esterification and chromatography.

In a first step, the synthesis method comprises an oxidation reactionwherein a polyethylene glycol compound is contacted with an oxidant fora sufficient amount time to produce the dicarboxylic acid analogue ofsaid polyethylene glycol compound. In some embodiments, the oxidationreaction is an oxoammonium-catalyzed oxidation reaction. In someembodiments, the oxidant is NaClO₂.

The synthesis method further comprises a subsequent step wherein theresulting dioic acid form of PEG is esterified to a lipid, theesterification reaction mediated by N-hydroxysuccinimide. In someembodiments, the resulting PEG-lipid is purified by chromatography.

The synthesis method further comprises a still further step wherein abisphosphonate-PEG-lipid is synthesized by an esterification reaction.In some embodiments, the esterification reaction is mediated byN-hydroxysuccinimide. In some embodiments, the bisphosphonate-PEG-lipidis purified by chromatography.

In some embodiments, the bisphosphonate-PEG-lipid is a compound ofFormula IV. In some embodiments, the bisphosphonate-PEG-lipid is acompound of Formula I. In some embodiments, the bisphosphonate-PEG-lipidis a compound of Formula Ia. In some embodiments, thebisphosphonate-PEG-lipid is the compound of Formula Ib.

Methods of Making PEG-Lipid Mixtures

In another aspect, the present disclosure provides a method of making aphospholipid composition capable of assembling containing a fluid havinga normal boiling point of less than 30° C. The method comprises thesteps of: (a) blending of PEG-lipids, including abisphosphonate-PEG-lipid, (b) homogenization, and (c) filtration.

In typical embodiments, step (a) comprises blending abisphosphonate-PEG-lipid, a compound of Formula II, and a compound ofFormula III.

In typical embodiments, the bisphosphonate-PEG-lipid is a compound ofFormula IV. In certain embodiments, the compound of Formula IV is acompound of Formula 1. In particular embodiments, the compound ofFormula I is a compound of Formula Ia. In particular embodiments, thecompound of Formula I is the compound of Formula Ib.

In some embodiments, the compound of Formula II is a compound of FormulaIIa. In some embodiments, the compound of Formula II is the compound ofFormula IIb. In some embodiments, compound of Formula III is a compoundof Formula IIIa.

In step (b), the blended mixture of PEG-lipids is homogenized.

In step (c), the homogenized mixture is filtered.

Method of Manufacturing Microspheres

In a further aspect, the present disclosure provides for a method ofmanufacturing microspheres, the method comprising (a) preparing aPEG-lipid mixture as set forth in the section above, the mixtureoptionally further comprising at least one pharmaceutically acceptableexcipient; and combining with water; (b) combining the PEG-lipid mixtureof step (a) in a vessel with a fluid having a normal boiling point ofless than 30° C.; and (c) agitating or otherwise energizing the vesselcomprising the PEG-lipid mixture and fluid from step (b) to obtainmicrospheres. In some embodiments, microspheres obtained from step (c)are provided for therapeutic use. In this case, the microspheres can bein a liquid composition. The liquid composition can be in a container.

In some embodiments, the method further comprises step (d), processingthe composition containing microspheres to lengthen its shelf life orexpand the range of environmental conditions for storage. In oneembodiment, step (d) involves lyophilizing the composition comprisingmicrospheres, but other methods known in the art can be used to lengthenshelf life of microsphere solution or expand the range of environmentalconditions for storage.

In some embodiments, the method further comprises filling thecomposition comprising the microspheres from step (c) or (d) intocontainers. In some embodiments, the method further comprises performingcertain of the processing steps in containers. In some embodiments, themethod further comprises the step of stoppering the containerscomprising the microspheres from step (c) or (d).

In some embodiments, the method further comprises aseptic processing.

In some embodiments, the phospholipid composition of step (a) furthercomprises a compound of Formula II or a salt thereof. In someembodiments, the phospholipid composition of step (a) further comprisesa compound of Formula III or a salt thereof. In some embodiments, thephospholipid composition of step (a) further comprises both a compoundof Formula II or a salt thereof and a compound of Formula III or a saltthereof.

In some embodiments, the compound of Formula I or a salt thereof used inthe method is a compound of Formula Ia, or a compound of Formula Ib. Insome embodiments, the compound of Formula II or a salt thereof used inthe method is a compound of Formula IIa or a compound of Formula IIb. Insome embodiments, the compound of Formula III or a salt thereof used inthe method is a compound of Formula IIIa.

In some embodiments, the phospholipid composition of step (a) isfiltered prior to step (b).

In some embodiments, the phospholipid composition is transferred to avessel after step (a) and before step (b). In some embodiments, some orall of steps (a)-(d) are performed by continuous processing.

In some embodiments, the lyophilized microspheres exist as lyophilizeddry powder or as water-free concentrate.

In some embodiments, the vessel may be stoppered while under vacuum.

In some embodiments, the lyophilized microspheres are more stable thanthe microsphere solutions. In some embodiments, the lyophilizedmicrospheres have longer shelf life than the microsphere solutions.

In some embodiments, the vessel is a unit dosage container.

In some embodiments, the fluid is n-decafluorobutane.

In some embodiments, the capped vessel is filled withn-decafluorobutane.

In some embodiments, the phospholipid composition of step (a) comprisesliposomes with a mean diameter from 40 nanometers to 5000 nanometers. Insome embodiments, the phospholipid composition of step (a) comprisesliposomes with a mean diameter from 100 nanometers to 1000 nanometers,200 nanometers to 900 nanometers, 300 nanometers to 800 nanometers, 400nanometers to 700 nanometers, 500 nanometers to 600 nanometers, 100nanometers to 200 nanometers, or 10 nanometers to 100 nanometers. Insome embodiments, the mean diameter of the liposomes in the phospholipidcomposition of step (a) is about 50 nanometers.

In some embodiments, the method manufactures energy-responsivemicrospheres. For example, the microspheres generated by the method cancollapse in response to application of energy. In some embodiments, theenergy is in the form of electromagnetic, acoustic, microwave, photonic,or other forms. The collapse of the microspheres can release energy,e.g., mechanical energy.

In some embodiments, the mean diameter of the microspheres beforelyophilization is about 0.1 microns to 1000 microns. In someembodiments, the mean diameter of the microspheres before lyophilizationis about 0.1 microns to 100 microns. In some embodiments, the meandiameter of the microspheres before lyophilization is about 0.1 micronsto 30 microns. In some embodiments, the mean diameter of themicrospheres before lyophilization is about 0.7 microns to 10 microns.In some embodiments, the mean diameter of the microspheres beforelyophilization is about 0.5 microns to 15.0 microns. In someembodiments, the mean diameter of the microspheres before lyophilizationis about 1.0 microns to 10.0 microns. In some embodiments, the meandiameter of the microspheres before lyophilization is about 1.0 micronsto 8.0 microns. In some embodiments, the mean diameter of themicrospheres before lyophilization is about 1.0 microns to 7.0 microns.In some embodiments, the mean diameter of the microspheres beforelyophilization is about 1.0 microns to 6.0 microns. In some embodiments,the mean diameter of the microspheres before lyophilization is about 1.0microns to 5.0 microns. In some embodiments, the mean diameter of themicrospheres before lyophilization is about 1.0 microns to 4.0. In someembodiments, the mean diameter of the microspheres before lyophilizationis about 1.0 microns to 3.0 microns. In some embodiments, the meandiameter of the microspheres before lyophilization is about 1.0 micronsto 2.0 microns.

In some embodiments, the method comprises step (d) involvinglyophilizing the composition comprising microspheres, and loss ofmicrospheres during lyophilization is no more than 50%. In someembodiments, the loss of the microspheres during lyophilization is nomore than 40%. In some embodiments, the loss of the microspheres duringlyophilization is no more than 30%. In another embodiment, the loss ofthe microspheres during lyophilization is no more than 25%. In someembodiments, the loss of the microspheres during lyophilization is nomore than 20%. In some embodiments, the loss of the microspheres duringlyophilization is no more than 15%. In some embodiments, the loss of themicrospheres during lyophilization is no more than 10%.

In some embodiments, the molecular weight of the compound of Formula Iis 1,500 to 5,000 Daltons. In some embodiments, the molecular weight ofthe compound of Formula I is 2,000 to 5,000 Daltons. In someembodiments, the molecular weight of the compound of Formula I is 2,000to 4,500 Daltons. In some embodiments, the molecular weight of thecompound of Formula I is 2,500 to 4,000 Daltons. In some embodiments,the molecular weight of the compound of Formula I is 3,000 to 3,500Daltons.

In some embodiments, the molecular weight of the compound of Formula IIis 1,500 to 5,000 Daltons. In some embodiments, the molecular weight ofthe compound of Formula II is 2,000 to 4,500 Daltons. In someembodiments, the molecular weight of the compound of Formula II is 2,500to 4,000 Daltons. In some embodiments, the molecular weight of thecompound of Formula II is 3,000 to 3,500 Daltons.

In some embodiments, the molecular weight of the compound of Formula IIIis 500 to 2,000 Daltons. In some embodiments, the molecular weight ofthe compound of Formula III is 750 to 1,750 Daltons. In someembodiments, the molecular weight of the compound of Formula III is1,000 to 1,500 Daltons. In some embodiments, the molecular weight of thecompound of Formula III is 1,100 to 1,400 Daltons. In some embodiments,the molecular weight of the compound of Formula III is 1200 to 1,300Daltons.

In some embodiments, the microspheres obtained from step (c) comprise0.01-5 mol % of a compound of Formula I. In some embodiments, themicrospheres comprise 0.05-5 mol % of a compound of Formula I. In someembodiments, the microspheres comprise 0.1-5 mol % of a compound ofFormula I. In some embodiments, the microspheres comprise 0.5-4.5 mol %of a compound of Formula I. In some embodiments, the microspherescomprise 1.0-4.0 mol % of a compound of Formula I. In some embodiments,the microspheres comprise 1.5-3.5 mol % of a compound of Formula I. Insome embodiments, the microspheres comprise 2.0-3.0 mol % of a compoundof Formula I.

In some embodiments, the microspheres obtained from step (c) comprise5-9.9 mol % of a compound of Formula II. In some embodiments, themicrospheres comprise 5.5-9.5 mol % of a compound of Formula II. In someembodiments, the microspheres comprise 6.0-9.0 mol % of a compound ofFormula II. In some embodiments, the microspheres comprise 6.5-8.5 mol %of a compound of Formula II. In some embodiments, the microspherescomprise 7.0-8.0 mol % of a compound of Formula II.

In some embodiments, the microspheres obtained from step (c) comprise80-95 mol % of the compound of Formula III. In some embodiments, themicrospheres comprise 82.5-92.5 mol % of the compound of Formula III. Insome embodiments, the microspheres comprise 85-90 mol % of the compoundof Formula III. In some embodiments, the microspheres comprise 87.5-92.5mol % of the compound of Formula III.

In some embodiments, the microspheres obtained from step (c) comprise nomore than 5 mol % of the compound of Formula I. In some embodiments, themicrospheres obtained from step (c) comprise no more than 4.5 mol % of acompound of Formula I. In some embodiments, the microspheres comprise nomore than 4.0 mol % of a compound of Formula I. In some embodiments, themicrospheres comprise no more than 3.5 mol % of a compound of Formula I.In some embodiments, the microspheres comprise no more than 3.0 mol % ofa compound of Formula I. In some embodiments, the microspheres compriseno more than 2.5 mol % of a compound of Formula I. In some embodiments,the microspheres comprise no more than 2.0 mol % of a compound ofFormula I. In some embodiments, the microspheres comprise no more than1.5 mol % of a compound of Formula I. In some embodiments, themicrospheres comprise no more than 1.0 mol % of a compound of Formula I.In some embodiments, the microspheres comprise no more than 0.5 mol % ofa compound of Formula I. In some embodiments, the microspheres compriseno more than 0.1 mol % of a compound of Formula I. In some embodiments,the microspheres comprise no more than 0.05 mol % of a compound ofFormula I.

In some embodiments, the microspheres obtained from step (c) comprise nomore than 10 mol % of the compound of Formula II. In some embodiments,the microspheres comprise no more than 9.5 mol % of a compound ofFormula II. In some embodiments, the microspheres comprise no more than9.0 mol % of a compound of Formula II. In some embodiments, themicrospheres comprise no more than 8.5 mol % of a compound of FormulaII. In another embodiment, the microspheres comprise no more than 8.0mol % of a compound of Formula II. In another embodiment, themicrospheres comprise no more than 7.5 mol % of a compound of FormulaII. In some embodiments, the microspheres comprise no more than 7.0 mol% of a compound of Formula II. In some embodiments, the microspherescomprise no more than 6.5 mol % of a compound of Formula II. In someembodiments, the microspheres comprise no more than 6.0 mol % of acompound of Formula II. In some embodiments, the microspheres compriseno more than 5.5 mol % of a compound of Formula II. In some embodiments,the microspheres comprise no more than 5.0 mol % of a compound ofFormula II.

In some embodiments, the microspheres obtained from step (c) comprise nomore than 95 mol % of the compound of Formula III. In some embodiments,the microspheres comprise no more than 92.5 mol % of the compound ofFormula III. In some embodiments, the microspheres comprise no more than90 mol % of the compound of Formula III. In some embodiments, themicrospheres comprise no more than 87.5 mol % of the compound of FormulaIII. In some embodiments, the microspheres comprise no more than 85.0mol % of the compound of Formula III. In some embodiments, themicrospheres comprise no more than 82.5 mol % of the compound of FormulaIII. In some embodiments, the microspheres comprise no more than 80.0mol % of the compound of Formula III.

In some embodiments, the average molecular weight of the compound ofFormula Ib in the microspheres of step (c) is about 3500 Daltons. Insome embodiments, the average molecular weight of the compound ofFormula Ib is about 3200 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula Ib is about 2500 Daltons. Insome embodiments, the average molecular weight of the compound ofFormula Ib is about 2000 Daltons.

In some embodiments, the average molecular weight of the compound ofFormula IIb in the microspheres of step (c) is about 3000 Daltons. Insome embodiments, the average molecular weight of the compound ofFormula IIb is about 2800 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula IIb is about 2600 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIb is about 2400 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula IIb is about 2200 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIb is about 2000 Daltons.

In some embodiments, the average molecular weight of the compound ofFormula IIIa in the microspheres of step (c) is about 840 Daltons. Insome embodiments, the average molecular weight of the compound ofFormula IIIa is about 790 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula IIIa is about 750 Daltons.In some embodiments, the average molecular weight of the compound ofFormula IIIa is about 700 Daltons. In some embodiments, the averagemolecular weight of the compound of Formula IIIa is about 650 Daltons.

In some embodiments, the microspheres obtained from step (c) comprise0.01-5 mol % of the compound of Formula Ib. In some embodiments, themicrospheres comprise 0.05-5 mol % of the compound of Formula Ib. Insome embodiments, the microspheres comprise 0.1-5 mol % of the compoundof Formula Ib. In some embodiments, the microspheres comprise 0.5-4.5mol % of a compound of Formula Ib. In some embodiments, the microspherescomprise 1.0-4.0 mol % of a compound of Formula Ib. In some embodiments,the microspheres comprise 1.5-3.5 mol % of a compound of Formula Ib. Insome embodiments, the microspheres comprise 2.0-3.0 mol % of a compoundof Formula Ib.

In some embodiments, the microspheres obtained from step (c) comprise5-9.9 mol % of the compound of Formula IIb. In some embodiments, themicrospheres comprise 6.0-9.0 mol % of a compound of Formula IIb. Insome embodiments, the microspheres comprise 6.5-8.5 mol % of a compoundof Formula IIb. In some embodiments, the microspheres comprise 7.0-8.0mol % of a compound of Formula IIb.

In some embodiments, the microspheres obtained from step (c) comprise80-95 mol % of the compound of Formula IIIa. In some embodiments, themicrospheres comprise 82.5-92.5 mol % of the compound of Formula IIIa.In some embodiments, the microspheres comprise 85-90 mol % of thecompound of Formula IIIa. In some embodiments, the microspheres comprise87.5-92.5 mol % of the compound of Formula IIIa.

In some embodiments, the microspheres obtained from step (c) comprise nomore than 5 mol % of the compound of Formula Ib. In some embodiments,the microspheres comprise no more than 4.5 mol % of a compound ofFormula Ib. In some embodiments, the microspheres comprise no more than4.0 mol % of a compound of Formula Ib. In some embodiments, themicrospheres comprise no more than 3.5 mol % of a compound of FormulaIb. In some embodiments, the microspheres comprise no more than 3.0 mol% of a compound of Formula Ib. In some embodiments, the microspherescomprise no more than 2.5 mol % of a compound of Formula Ib. In someembodiments, the microspheres comprise no more than 2.0 mol % of acompound of Formula Ib. In some embodiments, the microspheres compriseno more than 1.5 mol % of a compound of Formula Ib. In some embodiments,the microspheres comprise no more than 1.0 mol % of a compound ofFormula Ib. In some embodiments, the microspheres comprise no more than0.5 mol % of a compound of Formula Ib. In some embodiments, themicrospheres comprise no more than 0.1 mol % of a compound of FormulaIb. In some embodiments, the microspheres comprise no more than 0.05 mol% of a compound of Formula Ib.

In some embodiments, the microspheres obtained from step (c) comprise nomore than 10 mol % of the compound of Formula IIb. In some embodiments,the microspheres comprise no more than 9.5 mol % of a compound ofFormula IIb. In some embodiments, the microspheres comprise no more than9.0 mol % of a compound of Formula IIb. In some embodiments, themicrospheres comprise no more than 8.5 mol % of a compound of FormulaIIb. In some embodiments, the microspheres comprise no more than 8.0 mol% of a compound of Formula IIb. In some embodiments, the microspherescomprise no more than 7.5 mol % of a compound of Formula IIb. In someembodiments, the microspheres comprise no more than 7.0 mol % of acompound of Formula IIb. In some embodiments, the microspheres compriseno more than 6.5 mol % of a compound of Formula IIb. In someembodiments, the microspheres comprise no more than 6.0 mol % of acompound of Formula IIb. In some embodiments, the microspheres compriseno more than 5.5 mol % of a compound of Formula IIb. In someembodiments, the microspheres comprise no more than 5.0 mol % of acompound of Formula IIb.

In some embodiments, the microspheres obtained from step (c) comprise nomore than 95 mol % of the compound of Formula IIIa. In some embodiments,the microspheres comprise no more than 92.5 mol % of the compound ofFormula IIIa. In some embodiments, the microspheres comprise no morethan 90 mol % of the compound of Formula IIIa. In some embodiments, themicrospheres comprise no more than 87.5 mol % of the compound of FormulaIIIa. In some embodiments, the microspheres comprise no more than 85.0mol % of the compound of Formula Ma. In some embodiments, themicrospheres comprise no more than 82.5 mol % of the compound of FormulaIIIa. In some embodiments, the microspheres comprise no more than 80.0mol % of the compound of Formula IIIa.

In some embodiments, the molar ratio of the compound of Formula Ia tothe compound of Formula IIa in the microspheres obtained from step (c)ranges from 1:1000 to 1:1. In some embodiments of the microspheres, themolar ratio of the compound of Formula Ia to the compound of Formula IIaranges from 1:500 to 1:1. In some embodiments of the microspheres, themolar ratio of the compound of Formula Ia to the compound of Formula IIaranges from 1:100 to 1:1. In some embodiments, the molar ratio of thecompound of Formula Ia to the compound of Formula IIa ranges from 1:90to 1:5. In some embodiments, the molar ratio of the compound of FormulaIa to the compound of Formula IIa ranges from 1:80 to 1:10. In someembodiments, the molar ratio of the compound of Formula Ia to thecompound of Formula IIa ranges from 1:70 to 1:20. In some embodiments,the molar ratio of the compound of Formula Ia to the compound of FormulaIIa ranges from 1:60 to 1:30. In some embodiments, the molar ratio ofthe compound of Formula Ia to the compound of Formula IIa ranges from1:50 to 1:40.

In some embodiments of the pharmaceutical composition, the molar ratioof the compound of Formula IIa to the compound of Formula IIIa rangesfrom 1:20 to 1:8. In some embodiments, the molar ratio of the compoundof Formula IIa to the compound of Formula IIIa ranges from 1:18 to 1:10.In some embodiments, the molar ratio of the compound of Formula IIa tothe compound of Formula IIIa ranges from 1:16 to 1:12. In someembodiments, the molar ratio of the compound of Formula IIa to thecompound of Formula IIIa ranges from 1:15 to 1:13.

In some embodiments of the microspheres, the molar ratio of the compoundof Formula Ib to the compound of Formula IIb ranges from 1:1000 to 1:1.In some embodiments of the microspheres, the molar ratio of the compoundof Formula Ib to the compound of Formula IIb ranges from 1:500 to 1:1.In some embodiments of the microspheres, the molar ratio of the compoundof Formula Ib to the compound of Formula IIb ranges from 1:100 to 1:1.In some embodiments, the molar ratio of the compound of Formula Ib tothe compound of Formula IIb ranges from 1:90 to 1:5. In someembodiments, the molar ratio of the compound of Formula Ib to thecompound of Formula IIb ranges from 1:80 to 1:10. In some embodiments,the molar ratio of the compound of Formula Ib to the compound of FormulaIIb ranges from 1:70 to 1:20. In some embodiments, the molar ratio ofthe compound of Formula Ib to the compound of Formula IIb ranges from1:60 to 1:30. In some embodiments, the molar ratio of the compound ofFormula Ib to the compound of Formula IIb ranges from 1:50 to 1:40.

Unit Dosage Form

In a fourth aspect, the present disclosure provides for a unit dosagecontainer comprising a therapeutically effective amount of thepharmaceutical composition described herein.

In some embodiments, the unit dosage container comprises a phosphilipidcomposition, wherein the phospholipid composition comprises a compoundof Formula I, a compound of Formula II, and a compound of Formula III,and at least one pharmaceutically acceptable excipients. In someembodiments, the unit dosage container further comprises a fluid with anormal boiling point less than 30° C.

In some embodiments, the unit dosage container comprises microspheres,wherein the microspheres comprise a compound of Formula I, a compound ofFormula II, a compound of Formula III, a fluid with a normal boilingpoint less than 30° C., and at least one pharmaceutically acceptableexcipients. The microspheres can be obtained by the method describedherein related to the method of manufacturing microspheres.

In some embodiments of the unit dosage form, the compound of Formula Iis a compound of Formula Ia or Ib.

In some embodiments of the unit dosage form, the compound of Formula IIis a compound of Formula IIa or IIb.

In some embodiments of the unit dosage form, the compound of Formula IIIis a compound of Formula IIIa.

In some embodiments, the fluid with a normal boiling point of less than30° C. is a gas at body temperature. In some embodiments, the fluid isair, CO₂, sulfur hexafluoride, a fluorinated C₁₋₆ alkane, or acombination thereof.

In some embodiments, the fluid is n-decafluorobutane.

In some embodiments, the composition of the unit dosage containerfurther comprises trehalose and PLASDONE K12 as excipients. In someembodiments, the unit dosage container further comprises phosphatebuffered solution without calcium and magnesium ions.

In some embodiments, the composition of the unit dosage container iscapable of forming microspheres in the presence of water. In someembodiments, the composition of the unit dosage container is a liquidcomposition comprising microspheres.

In some embodiments, the microspheres of the unit dosage container existas lyophilized dry powder or as water-free concentrate. In someembodiments, the lyophilized dry powder is the lyophalate comprising ofmicrospheres. In some embodiments, the composition of the unit dosagecontainer is a product obtained by reconstitution of a lyophilizedformulation comprising microspheres.

In some embodiments, the residual water content of the lyophalatecomprising the microspheres is no more than 10% by weight. In someembodiments, the residual water content of the lyophalate comprising themicrospheres is no more than 9.5% by weight. In some embodiments, theresidual water content of the lyophalate comprising the microspheres isno more than 9.0% by weight. In some embodiments, the residual watercontent of the lyophalate comprising the microspheres is no more than8.5% by weight. In some embodiments, the residual water content of thelyophalate comprising the microspheres is no more than 8.0% by weight.In some embodiments, the residual water content of the lyophalatecomprising the microspheres is no more than 7.5% by weight. In someembodiments, the residual water content of the lyophalate comprising themicrospheres is no more than 7.0% by weight. In some embodiments, theresidual water content of the lyophalate comprising the microspheres isno more than 6.5% by weight. In some embodiments, the residual watercontent of the lyophalate comprising the microspheres is no more than6.0% by weight. In some embodiments, the residual water content of thelyophalate comprising the microspheres is no more than 5.5% by weight.In some embodiments, the residual water content of the lyophalatecomprising the microspheres is no more than 5.0% by weight. In someembodiments, the residual water content of the lyophalate comprising themicrospheres is no more than 4.5% by weight. In some embodiments, theresidual water content of the lyophalate comprising the microspheres isno more than 4.0% by weight. In some embodiments, the residual watercontent of the lyophalate comprising the microspheres is no more than3.5% by weight. In some embodiments, the residual water content of thelyophalate comprising the microspheres is no more than 3.0% by weight.In some embodiments, the residual water content of the lyophalatecomprising the microspheres is no more than 2.5% by weight. In someembodiments, the residual water content of the lyophalate comprising themicrospheres is no more than 2.0% by weight. In some embodiments, theresidual water content of the lyophalate comprising the microspheres isno more than 1.5% by weight. In some embodiments, the residual watercontent of the lyophalate comprising the microspheres is no more than1.0% by weight. In some embodiments, the residual water content of thelyophalate comprising the microspheres is no more than 0.5% by weight.In some embodiments, the residual water content of the lyophalatecomprising the microspheres is no more than 0.4% by weight. In someembodiments, the residual water content of the lyophalate comprising themicrospheres is no more than 0.3% by weight. In some embodiments, theresidual water content of the lyophalate comprising the microspheres isno more than 0.2% by weight. In some embodiments, the residual watercontent of the lyophalate comprising the microspheres is no more than0.1% by weight. In some embodiments, the residual water content of thelyophalate comprising the microspheres is no more than 0.01% by weight.

In some embodiments, the composition of the unit dosage container isprepared by any one of the methods described herein.

In some embodiments, the unit dosage container is sealed by a crimp-topcap fitted with a septum. In some embodiments, the unit dosage containeris airtight.

Kits

In another aspect, the present disclosure provides for a kit comprisingat least one unit dosage container as described herein and instructionsfor using said kit.

In some embodiments, the composition within each of the at least oneunit dosage container is capable of forming microspheres in the presenceof water.

In some embodiments of the kit, the unit dosage container furthercomprises trehalose and PLASDONE K12 as pharmaceutically acceptableexcipients.

In some embodiments, the kit further comprises a container comprising anaqueous solution. In some embodiments, the aqueous solution is sterilewater. In some embodiments, the aqueous solution is a saline solutionready for perfusion.

In some embodiments, the kit further comprises a syringe. In someembodiments, the kit further comprises a needle. In some embodiments,the kit further comprises a needle-free syringe (e.g., Mini-Spike®)comprising a sharp tip, wherein the needle-free syringe is capable ofpiercing through a stopper.

In some embodiments, the kit further comprises a container of fluid,wherein the fluid is air, nitrogen, argon, CO₂, sulfur hexafluoride, afluorinated C₁₋₆ alkane, or a combination thereof. In some embodiments,the container is a syringe.

In some embodiments of the kit, the fluorinated C₁₋₆ alkane is selectedfrom octafluoropropane, n-decafluorobutane, and dodecafluoropentane.

In some embodiments of the kit, the fluid is n-decafluorobutane.

In some embodiments, the kit further comprises a gel pad or ultrasoundgel.

In some embodiments, the kit further comprises an apparatus selectedfrom Mix2Vial® apparatus and a vented vial adapter.

Method of Reconstitution

In yet another aspect, the present disclosure provides for a method ofreconstituting the microspheres as prepared according to any one of themethods as described herein. In some embodiments, lyophalate comprisingof microspheres are reconstituted. In some embodiments, the method ofreconstitution comprises adding a sufficient amount of sterile water ora saline solution to the microsphere solution described in the abovesections in a container. The method can further comprise adding a volumeof gas to the container and shaking the container. Specifically, in someembodiments, the method of reconstituting microspheres comprises:

-   -   (a) adding a sufficient amount of sterile water or a saline        solution to the microspheres inside the container;    -   (b) optionally adding a volume of gas to the container of step        (a); and    -   (c) optionally shaking the container of step (b).

In some embodiments of the method of reconstitution, the amount ofsterile water or the saline solution is no more than 1000 milliliters.In some embodiments of the method of reconstitution, the amount ofsterile water or the saline solution is no more than 750 milliliters. Insome embodiments of the method of reconstitution, the amount of sterilewater or the saline solution is no more than 500 milliliters. In someembodiments of the method of reconstitution, the amount of sterile wateror the saline solution is no more than 200 milliliters. In someembodiments of the method of reconstitution, the amount of sterile wateror the saline solution is no more than 100 milliliters. In someembodiments, the amount of sterile water or the saline solution is nomore than 90 milliliters. In some embodiments, the amount of sterilewater or the saline solution is no more than 80 milliliters. In someembodiments, the amount of sterile water or the saline solution is nomore than 70 milliliters. In some embodiments, the amount of sterilewater or the saline solution is no more than 60 milliliters. In someembodiments, the amount of sterile water or the saline solution is nomore than 50 milliliters. In some embodiments, the amount of sterilewater or the saline solution is no more than 40 milliliters. In someembodiments, the amount of sterile water or the saline solution is nomore than 30 milliliters. In some embodiments, the amount of sterilewater or the saline solution is no more than 20 milliliters. In someembodiments, the amount of sterile water or the saline solution is nomore than 10 milliliters. In some embodiments, the amount of sterilewater or the saline solution is no more than 5 milliliters. In someembodiments, the amount of sterile water or the saline solution is nomore than 1 milliliters. In some embodiments, the amount of sterilewater or the saline solution is no more than 0.5 milliliters. In someembodiments, the amount of sterile water or the saline solution is nomore than 0.3 milliliters.

In some embodiments, the amount of the sterile water or the salinesolution added is sufficient to produce a homogeneous mixture comprisingthe reconstituted microspheres.

In some embodiments of the method of reconstitution, the shaking of thecontainer lasts no more than 180 seconds. In some embodiments, theshaking of the container lasts no more than 160 seconds. In someembodiments, the shaking of the container lasts no more than 140seconds. In some embodiments, the shaking of the container lasts no morethan 120 seconds. In some embodiments, the shaking of the containerlasts no more than 100 seconds. In some embodiments, the shaking of thecontainer lasts no more than 80 seconds. In some embodiments, theshaking of the container lasts no more than 60 seconds. In someembodiments, the shaking of the container lasts no more than 40 seconds.In some embodiments, the shaking of the container lasts no more than 20seconds. In some embodiments, the shaking of the container lasts no morethan 10 seconds. In some embodiments, the shaking of the container lastsno more than 5 seconds.

In some embodiments of the method of reconstitution, the shaking of thecontainer lasts no more than 160 seconds. In some embodiments, theshaking of the container lasts at least 140 seconds. In someembodiments, the shaking of the container lasts at least 120 seconds. Insome embodiments, the shaking of the container lasts at least 100seconds. In some embodiments, the shaking of the container lasts atleast 80 seconds. In some embodiments, the shaking of the containerlasts at least 60 seconds. In some embodiments, the shaking of thecontainer lasts at least 40 seconds. In some embodiments, the shaking ofthe container lasts at least 20 seconds. In some embodiments, theshaking of the container lasts at least 10 seconds. In some embodiments,the shaking of the container lasts at least 5 seconds.

Method of Treatment

In a final aspect, the present disclosure provides for a method oftreating a medical condition involving an abnormal or obstructive mass.In some embodiments, the medical condition involves kidney stones,urinary stones, biliary stones, blood clots, fibroids, cancerous tumors,and atheromatous plaques. In some embodiments, the subject hasurolithiasis.

In some embodiments, the method comprises: administering to a subjectwith the medical condition an effective amount of the reconstitutedmicrosphere solution as described herein so as to bring the microspheresinto contact with the abnormal or obstructive mass (e.g., urinary stone,kidney stone, biliary stones, blood clots, fibroids, cancerous tumors,and atheromatous plaques); and directionally applying an energy, at afrequency that excites the fluid within the microspheres, to theabnormal or obstructive mass within the subject.

In some embodiments, the reconstituted microsphere solution isadministered into the ureter of the subject through a urinary catheter.

In some embodiments, the applied energy is in the form ofelectromagnetic, acoustic, microwave, photonic, laser, or other forms.

In some embodiments, the applied energy is ultrasonic. In someembodiments, the applied energy is acoustic energy.

In some embodiments, the acoustic energy is ultrasonic energy in thefrequency range from 100 kilohertz (kHz) to 2 megahertz (MHz). In someembodiments, the acoustic energy is ultrasonic energy in the frequencyrange from 200 kilohertz (kHz) to 1.5 megahertz (MHz). In someembodiments, the acoustic energy is ultrasonic energy in the frequencyrange from 300 kilohertz (kHz) to 1.2 megahertz (MHz). In someembodiments, the acoustic energy is ultrasonic energy in the frequencyrange from 500 kilohertz (kHz) to 1 megahertz (MHz).

In some embodiments, the applied ultrasonic energy is associated withpeak pressures in the range 0.1 MPa to 10 MPa. In some embodiments, theapplied ultrasonic energy is associated with peak pressures in the range1 MPa to 10 MPa. In some embodiments, the applied ultrasonic energy isassociated with peak pressures in the range 2 MPa to 9 MPa. In someembodiments, the applied ultrasonic energy is associated with peakpressures in the range 4 MPa to 8 MPa. In some embodiments, the appliedultrasonic energy is associated with peak pressures in the range 5 MPato 7 MPa.

In some embodiments, the applied energy is generated with intraluminalenergy from solid-state, pulsed laser.

In some embodiments, the wavelength of the laser energy is in the range1000 nm to 2500 nm. In some embodiments, the wavelength of the laserenergy is in the range 2050 nm to 2150 nm. In some embodiments, thevaporization of a liquid phase by the laser energy is associated with amoving phase boundary that produces pressure effects in the liquidphase.

In some embodiments, the laser energy has a frequency in the rangebetween 1 kHz to 1 MHz. In some embodiments, the laser energy has afrequency in the range between 1 kHz to 1 MHz, between 10 kHz to 500kHz, between 50 kHz to 100 kHz, or between 10 kHz to 50 kHz.

In some embodiments, the energy is applied for a sufficient amount oftime to fragment the urinary stone.

In some embodiments, the amount of time of the applied energy is no morethan 100 minutes. In some embodiments, the amount of time of the appliedenergy is no more than 90 minutes. In some embodiments, the amount oftime of the applied energy is no more than 80 minutes. In someembodiments, the amount of time of the applied energy is no more than 70minutes. In some embodiments, the amount of time of the applied energyis no more than 60 minutes. In some embodiments, the amount of time ofthe applied energy is no more than 50 minutes. In some embodiments, theamount of time of the applied energy is no more than 40 minutes. In someembodiments, the amount of time of the applied energy is no more than 30minutes. In some embodiments, the amount of time of the applied energyis no more than 25 minutes. In some embodiments, the amount of time ofthe applied energy is no more than 20 minutes. In some embodiments, theamount of time of the applied energy is no more than 15 minutes. In someembodiments, the amount of time of the applied energy is no more than 10minutes.

In some embodiments, the applied photonic energy causes a change involume of the microspheres or other cavitation effects of themicrospheres.

In some embodiments, the cavitation of the microspheres causes pressuregradient changes and mechanical effects in the urinary stone around thereconstituted microspheres.

In some embodiments, the pressure gradient changes and other mechanicaleffects are capable of fragmenting urinary stones.

In some embodiments, the subject is human. In some embodiments, thesubject is an animal.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the present technology and are not to be construed in any wayas limiting the scope of the present technology.

Unless otherwise stated, all temperatures are in degrees Celsius.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should be allowed for.

Any methods that are functionally equivalent are within the scope of thepresent technology. Various modifications of the present technology inaddition to those described herein will become apparent to those skilledin the art from the foregoing description and accompanying figures. Suchmodifications fall within the scope of the appended claims.

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined, it has its generallyaccepted meaning.

-   -   aq.=aqueous    -   LC-MS=liquid chromatography-mass spectrometry    -   MS=mass spectrometry    -   THF=tetrahydrofuran    -   NaHCO₃=sodium bicarbonate    -   DIEA=diisopropylethylamine    -   MS=mass spectrometry    -   NaH=sodium hydride    -   o/n=overnight    -   HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-trI        zolo[4,5-b]pyridinium 3-oxid hexafluorophosphate    -   r.t.=room temperature    -   LAH=lithium aluminum hydride    -   DCM=dichloromethane    -   DMF=dimethylformamide    -   DMSO=dimethyl sulfoxide    -   equiv.=equivalent    -   EtOAc=ethyl acetate    -   EtOH=ethanol    -   gram    -   hours    -   HCl=hydrochloric acid    -   HPLC=high-performance liquid chromatography    -   HOAc=acetic acid    -   molar    -   MeOH=methanol    -   mg=milligrams    -   mL=milliliters    -   mmol=millimols    -   mp=melting point    -   m/z=mass to charge ratio    -   NaCl=sodium chloride    -   Na₂CO₃=sodium carbonate    -   NMR=nuclear magnetic resonance    -   NaOH=sodium hydroxide    -   Na₂SO₄=sodium sulfate    -   NHS=n-hydroxysuccinimide    -   ppm=parts per million    -   TLC=thin layer chromatography    -   UV=ultraviolet    -   wt %=weight percent    -   μM=micromolar

General Experimental Details

Final compounds and phospholipid composition were confirmed by NMR.¹H-NMR, ³¹P-NMR, ¹³C-NMR spectra were recorded in CDCl₃ (residualinternal standard CHCl₃=δ 7.26), DMSO-d₆ (residual internal standardCD₃SOCD₂H=δ 2.50), methanol-d₄ (residual internal standard CD₂HOD=δ3.20), or acetone-d₆ (residual internal standard CD₃COCD₂H=δ 2.05), or amixture of CDCl₃ and methanol-d₄. The chemical shifts (δ) reported aregiven in parts per million (ppm) and the coupling constants (J) are inHertz (Hz). The spin multiplicities are reported as s=singlet, bs=broadsinglet, bm=broad multiplet, d=doublet, t=triplet, q=quartet,p=pentuplet, dd=doublet of doublet, ddd=doublet of doublet of doublet,dt=doublet of triplet, td=triplet of doublet, tt=triplet of triplet, andm=multiplet.

Example 1: Synthesis of the Compound of Formula Ib Step 1—Synthesis ofPEG2DA

PEG2000 was dissolved in acetonitrile and warmed to 40° C. Followingdissolution, an aqueous potassium phosphate buffer adjusted to pH ˜7.5with phosphoric acid is charged, followed by the addition of TEMPO.Aqueous solutions of sodium chlorite and sodium hypochlorite are thensimultaneously charged to the reaction mixture over the course of 30minutes to 1 hour. When the charge is complete, the reaction is stirredat 40° C. for a minimum of 24-48 hours. After the minimum stir time, thereaction is assayed for completion by TLC analysis. When deemedcomplete, the mixture is cooled to 0° C. and quenched with an aqueoussodium thiosulfate solution. The quenched solution is then acidifiedwith aqueous hydrochloric acid to pH<3 and the organic material isextracted with chloroform/methanol/water partitions. Solvent is thenremoved from the organic phase via rotary evaporation and the materialis dried with toluene cycles. The crude material is then purified byprecipitation from toluene with diethyl ether. FIG. 8 shows the ¹H-NMRspectrum of purified PEG2DA.

Step 2—Synthesis of DSPE-PEG2-COOH

PEG2DA was dissolved in toluene and warmed to 90° C. CDI was addedportion-wise to control carbon dioxide evolution. Once all solids weredissolved and all bubbling has ceased, solid DSPE was added to thereaction mixture, followed by triethylamine. The mixture was stirred at90° C. for 12-24 hours, at which time reaction completion was checked byTLC. Once complete, the reaction mixture was quenched with 5-10 mL ofmethanol and cooled to ambient temperature. Once cooled, the solutionwas diluted with chloroform and partitioned against 1 M aqueoushydrochloric acid to remove imidazole, followed by a brine wash. Themixture was neutralized with ammonium hydroxide prior to bulk solventremoval via rotary evaporation. The crude mixture was then sequentiallychromatographed first with normal phase media(chloroform/methanol/ammonium hydroxide gradient) then with reversephase media (methanol/water gradient) including a column wash withsodium chloride to achieve the sodium salt of DSPE-PEG2—COOH. FIGS. 9and 10 show the ¹H-NMR and ³¹P-NMR spectra of DSPE-PEG2-COOHrespectively.

Step 3—Synthesis of the Compound of Formula Ib

Prior to reaction, alendronic acid was dissolved in tetrabutylammoniumhydroxide (40 wt % solution in water) and the water was removedazeotropically using toluene, followed by vacuum drying. Followingalendronate-TBA preparation, DSPE-PEG2-COOH was dissolved in a mixtureof 85:15 anhydrous chloroform/anhydrous dimethylformamide and warmed to40° C. To the solution is added N-Hydroxysuccinimide (NHS), followed by1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), and the mixturewas stirred at 40° C. for 4-6 hours. After the allotted time, ¹H NMRspectroscopy was used to assay for DSPE-PEG2-COO—NHS ester formation(typical reaction completion was >75% by ¹H-NMR analysis). Once DSPEPEG2 COO—NHS ester formation was confirmed, the previously preparedalendronate-TBA salt was dissolved in anhydrous chloroform and addeddirectly to the reaction vessel, followed by triethylamine. The reactiontemperature was then increased to 50° C. and allowed to stir attemperature for 18-24 hours, at which time reaction completion wasassayed by TLC. Once complete, the reaction mixture was partitionedthree times against 1 M aqueous hydrochloric acid to remove excessalendronate, followed by two brine washes to remove excess acid. Themixture was neutralized with ammonium hydroxide prior to bulk solventremoval via rotary evaporation. The material was then treated withDowex® 50WX8 hydrogen form ion exchange resin to remove bulktetrabutylammonium counterion. The resin was filtered and bulk solventwas removed from the filtrate, then the material was chromatographedwith reverse phase media (methanol/water gradient) including a columnwash with ammonium chloride to achieve the the compound of Formula Ib.FIG. 11 and FIG. 12 show the ¹H-NMR and ³¹P-NMR spectra of the compoundof Formula Ib respectively, with the structure:

The method synthesizing of the compound of Formula Ib described hereinis summarized below:

Example 2: Synthesis of Microspheres

The compound of Formula Ib generated as described in Example 1 was mixedwith other lipid compositions—

DSPE-PEG2k (the compound of Formula IIb)

and

DSPC (the compound of Formula IIIa)

and water. ³¹P-NMR spectrum of the mixture (FIG. 14) shows three peaks,each corresponding to each lipid composition, the compound of FormulaIb, DSPC, or DSPE-PEG2k. Signals for each peak indicate relative amountsof the compound of Formula Ib (28.37 for “DSPE PEG Alendronate”), DSPC(1363.68 for “DSPC”), and DSPE-PEG2k (107.55 for “DSPE PEG”) in themixture.

A liquid phase of the mixture comprising the compound of Formula Ib,DSPC, and DSPE-PEG2k was processed with a homogenizer with multiplepasses until the desired diameter and PDI was achieved. Samples fromeach pass was analyzed for size distribution using dynamic lightscattering (DLS) and the result is provided in FIG. 4. The formulationis diluted in phosphate buffered saline and analyzed with a Mobius(Wyatt).

n-decafluorobutane (C₄F₁₀) was added to the lipid mixture and themixture was agitated for 1-2 minutes. Images of the lipid mixture before(left) and after (right) the steps of n-decafluorobutane addition andagitation are provided and compared in FIG. 5. The images illustratequalitative differences of the formulations.

The Beckman Coulter Multisizer 4e with a 30 μm aperture was used tocharacterize microspheres for particle number density and particlediameter by electro-zone sensing. Sample volume ranged from 1 to 10 μLin 100 mL of IsotonII diluent. An exemplary result is provided in FIG.7. The results show that microspheres with average sizes ranging between0.7 and 2 μm were generated.

Example 3: Accumulation Assay

The microspheres generated as described in Examples 1 and 2 werecharacterized with the Beckman Coulter Multisizer 4e in a time coursestudy while incubated with hydroxyapatite beads. Microspheres wereanalyzed by the Multisizer for particle density at various time points.The change in concentration of microspheres was used to calculate thenumber of microspheres accumulating on the surface of hydroxyapatitebeads. A control microsphere sample without the compound of Formula Ibwas also analyzed.

As summarized in FIG. 6, the experiment demonstrated significantaccumulation of microspheres with the compound of Formula Ib ontohydroxyapatite beads over the time course study. The results suggestthat microspheres comprising the compound of Formula Ib have a highaffinity to hydroxyapatite beads.

Example 4: Headspace Analysis

The headspace sample was pulled directly from the container containing apharmaceutical composition of the compound of Formula Ib andn-decafluorobutane using a manual gas chromatography syringe. Theheadspace sample was immediately injected into the GC. GC signals fromthe experiment is provided in FIG. 13. It shows a peak indicatingn-decafluorobutane.

Example 5: Use of Microspheres with an External Acoustic Energy Sourcefor Treatment of Urolithiasis

Microspheres generated as described in Examples 1 and 2 wereadministered via a cystoscopically positioned 5 Fr catheter to patientsdiagnosed with urinary stones. Patients had presented with acute renalcolic and had a diagnosis of stone disease confirmed by CT scan. Thecystoscope was removed after positioning of the catheter in the affectedside of the patient's upper urinary tract and a syringe containing amicrosphere solution connected to the distal end of the catheter.0.5-1.0 mL volumes of microsphere solution were injected at multipleintervals over the course of procedures ranging in duration from 20 to90 minutes. Acoustic energy was applied at multiple intervals over thecourse of the procedure. The acoustic energy source used, a SmartSphereconsole with a treatment head designed to be held against the skin of apatient's lower back or inguinal region, was engineered to interact withmicrospheres on or near stone surfaces, bringing about stone erosion,pitting and fragmentation, and had been shown to be safe in extensivestudies in porcine models. Many urinary stone patients have beensuccessfully treated using this method.

EQUIVALENTS AND INCORPORATION BY REFERENCE

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

The invention claimed is:
 1. A compound of Formula Ia:

or a salt, isomer, or salt of an isomer thereof.
 2. A compound ofFormula Ib: